

PRESENTED BY 













































































































































































































































































































































































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UNIVERSITY OF PENNSYLVANIA 


CHROMOSOME NUMBER AND PAIRS 
IN THE SOMATIC MITOSES OF 
AMBYSTOMA TIGRINUM 


BY 

ip* i 

CHARLES L. PARMENTER 


A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL IN 
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR 
THE DEGREE OF DOCTOR OF PHILOSOPHY 


Reprinted from the 

Journal of Morphology, Volume 33, Number 1, December, 1919 
Philadelphia 



tt> c t3 
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Gift 

University 
f£B IS ($20 


AUTHOR’S AB3TRACT OF TH13 PAPER ISSUED 
BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 13 


Reprinted from Journal of Morphology 
Vol. 33, No. 1, December, 1919 


CHROMOSOME NUMBER AND PAIRS IN THE 
SOMATIC MITOSES OF AMBYSTOMA 1 
TIGRINUM 

CHARLES L. PARMENTER 
Zoological Laboratory , University of Pennsylvania 
THIRTY-SEVEN FIGURES (NINE PLATES) 

CONTENTS 

Introduction. 169 

Technique. 171 

Observations. 176 

A. The number of chromosomes. 176 

a. Method of determining the number. 177 

1. Procedure. 177 

2. Clearness and classification of the complexes. 178 

b. Possible variation in number in uncounted complexes. 181 

c. Abnormal complexes_\.;. 182 

B. Somatic chromosome pairs. 184 

а. Introductory statement. 184 

б. Mensuration. 185 

1. Types of cells. 186 

2. Method. 186 

3. Sources of error. 186 

c. Results of measurements. 191 

1. Criteria for determining pairs. 191 

2. Evidence for the existence of pairs. 194 

3. Summary. 199 

Discussion. 200 

A. Introductory statement. 200 

B. Constancy of chromosome number. 200 

C. Variations in other Urodeles. 206 

D. Variations in other forms. 206 

E. Fragmentation. 207 

F. Existence of pairs. 208 

a. Pairs in the germ cells. 208 

b. Pairs in the somatic cells. 210 

1. Meves’ measurements. 211 

2. Della Valle’s measurements. 215 

3. Results in Ambystoma tigrinum. 216 

c. Constant relative size relations. 220 

d. Summary of measurements. 220 

Summary of conclusions. 221 


1 Also known as Amblystoma. 

169 





































170 


CHARLES L. PARMENTER 


INTRODUCTION 

It is believed (McClung, ’17, pp. 536-38) that the chromatin 
of an organism is, for the most part at least, the idioplasm, and 
consists of a definite linearly arranged series of differentiated 
materials which is perpetuated from generation to generation. 
The chromosomes which are essentially constant in number in 
an individual are thought to constitute the visible mechanism 
for this perpetuation. This conception is known as the theory 
of the individuality of the chromosomes, which is quite generally 
accepted by all who have an intimate acquaintance with chro¬ 
mosome behavior. However, there are a few not so acquainted 
who strenuously oppose the theory. 

Among these is Della Valle (’09, ’ll, ’12), who presents some 
data and a large amount of discussion in an effort to disprove 
this theory upon the claim that the chromosome number in an 
individual is not constant, but is simply the quotient of the 
quantity of chromatin divided by the average size of the chro¬ 
mosomes. This removes from them any constancy of organiza¬ 
tion and contradicts the above theory. These observations have 
been cited by other opponents of the theory as cytological 
evidence in favor of their contentions. Della Valle’s conclusions 
are based upon observations made upon dividing cells of the 
peritoneum and blood-cells of Salamandra maculosa, together 
with a large amount of data taken from the observations of 
others. 

Meves (’ll) and Della Valle (’12) further oppose the theory 
upon the basis of linear measurements made upon the spermat- 
ogonial and somatic chromosomes of Salamandra maculosa in 
denying Montgomery’s (’01) and Sutton’s (’02) claim that the 
chromosomes occur in pairs whose homologues are of equal 
length, and that approximately constant size relations among 
chromosomes are maintained from one cell generation to 
another. 

In the spring of 1916 I was fortunate in obtaining peritoneal 
and other somatic tissues of Ambystoma tigrinum. This made 
it possible to repeat Della Valle’s observations upon the somatic 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 171 


cells of the same and other tissues of this closely related species 
and thus to determine whether such a variation as he claims is 
present in the somatic tissues of other Amphibians. Also the 
chromosomes of some cells in my material are sufficiently 
favorable for measurements to permit a reconsideration of their 
length relationships. 

Since this paper is regrettably controversial, it is necessary to 
give careful attention to all the methods and conditions under 
which the preparations and the observations were made. Della 
Valle also lays much emphasis upon this point, and therefore 
considerable space is devoted to this problem. 

For’facilities in collecting and preparing this material I am 
indebted to the courtesy of the Department of Zoology of the 
University of Minnesota, and to Prof. C. P. Sigerfoos I owe the 
loan of several very excellent preparations. The work was done 
under the direction of Prof. C. E. McClung, of the University 
of Pennsylvania, toward whom I feel especially grateful for 
constant encouragement and valuable criticism, and for his 
characteristically generous and kindly interest at all times. I 
am also greatly indebted to other members of the department, 
especially to Dr. Eleanor Carothers and Dr. D. H. Wenrich, for 
helpful suggestions and very painstaking criticisms. 

TECHNIQUE 

The material used was obtained during the spring of 1916 
from larvae of Ambystoma tigrinum, which were abundant in 
the ponds and lagoons near the College of Agriculture of the 
University of Minnesota. Mitotic figures in epithelial cells of 
the tail, gill plates, and lung, and of the endothelium from 
peritoneum and mesentery were studied. 

Tail epithelium 

Very excellent preparations of this tissue were kindly loaned 
me by Prof. Charles P. Sigerfoos, of the University of Minnesota. 
These were made from the tails of larvae f to 1| inches in length 
obtained during the last of May and the first of June during 
several years. 


172 


CHARLES L. PARMENTER 


The living larvae were thrown into Flemming’s stronger solu¬ 
tion. After about four hours of fixation, the tails were split 
dorsoventrally 2 into two thin plates of cells. These two plates 
of cells were then fixed twenty hours longer. After washing 
in running tap-water for twelve hours or more, the pieces were 
stained in toto in Heidenhain’s haematoxylin, carefully dehy¬ 
drated, and cleared in xylol and mounted in damar. 

Gill-plate epithelium 

The most successful gill-plate preparations were also obtained 
from larvae f to If inches long. In each larva there are eight 
gill plates, one subtended from each gill arch and another behind 
each posterior gill cleft. These gill plates contain very numer¬ 
ous mitotic figures. They are composed of two epithelial 
lamellae with connective-tissue cells and capillaries lying between 
them. The two layers, unseparated, are so thin that they give 
very excellent preparations. 

The material was fixed in situ by dropping the living larvae 
into the Flemming’s stronger solution as soon as they were taken 
from the net. They were fixed in situ twenty-four hours and 

2 Haecker (’99) describes a very successful method of separating these two plates 
of cells. The posterior end of the larva is cut off after fixation just in front of 
the cloaca. With a sharp scalpel the thick cephalic end of the tail is split dorso¬ 
ventrally through the middle of the vertebra to a depth of an eighth of an inch 
or more. By grasping with the forceps the ends thus made free, the two layers 
of epithelium can be pulled apart in a manner similar to separating two sheets 
of fly-paper with adhesive surfaces sticking together. Professor Sigerfoos advises 
separating the two layers after about four hours of fixation and then allowing to 
fix about twenty hours longer. 

The numerous large mitotic figures in various stages with clear cell walls 
which can be studied without an immersion lens makes this material excellent 
for the class-room. The gill-plate preparations are equal or superior to those 
of the tail epithelium. Those of larger larvae are too thick when mounted in 
toto, but give very satisfactory preparations when separated. Peritoneal prepa¬ 
rations of larger larvae contain fewer mitoses and the cell walls are indistinct. 
However, preparations can be made from the more rapidly growing shorter 
larvae from which the gill-plates were taken and would probably contain more 
divisions. Preparations of Ambystoma punctatum are less favorable than those 
of A. tigrinum because there are fewer figures, more pigment cells in the tail 
epithelium and the gill-plates are small and thicker. 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 173 


washed in running tap-water. The larvae from which the gill- 
plates were taken were fixed for other purposes, and no special 
effort was made to insure good fixation of the plates. They 
were preserved in position in 5 per cent formalin, which was 
later gradually replaced with 80 per cent alcohol. 

The fixed gill-plates were carefully removed from the larvae in 
80 per cent alcohol and were left attached to the gill arches, 
which, in subsequent handling, were grasped by forceps to prevent 
injury of the plates. Hydrogen peroxide was added to the 80 
per cent alcohol drop by drop through a fine glass capillary 
siphon until the solution amounted to equal parts of each. 
In this the plates were bleached for four to twelve hours and then 
transferred to the mordant by the above-mentioned drop-process 
and stained in iron haematoxylin. They were dehydrated by 
this drop method, cleared in cedar-wood oil followed by xylol, 
cut from gill arches, after being transferred to the slide, and 
then covered with damar and a thin cover-glass. While the 
damar was hardening they were kept for twenty-four hours or 
more under slight pressure to insure flat preparations. 

The peritoneum , mesentery , and lungs 

These preparations were made from larvae 3 to 4 inches long. 
All the tissues of a given individual were not only fixed together 
in the same fixative for the same length of time, but also received 
the same treatment in all subsequent processes. They were put 
into the fixatives within an estimated maximum of two minutes 
after the first incision. Two methods of procedure were used in 
preparing these tissues for fixation: 

1. In order to avoid any possible unfavorable effect of cap¬ 
tivity, the tissues were fixed in situ in the field as soon as the 
larvae were taken from the net. The animals were prepared for 
fixation as follows: With sharp scissors the body wall was cut 
open along the mid ventral line and also lateral incisions were 
made on each side at right angles to the first incision behind the 
pectoral girdle and in front of the pelvic girdle, so that the two 
halves of the bcdy wall fell away from the viscera and opened 


174 


CHARLES L. PARMENTER 


wide the body cavity. The folds of the viscera were pulled 
apart and the whole larva was plunged into the fixative. This 
secured immediate and uniform fixation. The operation requires 
less than a minute and the incisions are apparently painless, for 
the larva does not often struggle. 

2. The body walls, lungs, and viscera were removed from the 
body of the larvae before fixing, either in the field or at the 
laboratory. The peritoneum was fixed in situ on the body walls. 
Only normally inflated lungs were used, and these were ligated 
anteriorly before removal from the body to prevent them from 
collapsing. After fixing one or two hours, they were cut into 
two or more flat longitudinal strips and returned to the fixative. 
The mesentery, attached to the intestine, was spread out flat 
on a piece of glass and the whole immersed in the fixative with 
the tissue beneath. 

Fixatives 

The fixatives used were: 1) Flemming’s stronger solution, 
thirty hours; 2) Bouin’s solution, forty-three hours; 3) Bouin’s 
solution, to which was added 1^ grams of chromic acid crystals 
per 100 cc., twenty to twenty-four hours; 4) Hermann’s solution 
with two parts of osmic acid (Lee, ’13, p. 38) twelve to eighteen 
hours; 5) a solution of saturated picric acid 75 cc., formalin 15 
cc., glacial acetic acid 10 cc., urea crystals 2 grams, thirty to 
forty-three hours. The urea should be added gradually to the 
solution warmed to about 40°C., otherwise a precipitate is 
formed. 

It is a difficult matter to decide which solution gave the best 
fixation. The prettiest cells were fixed in Hermann’s and the 
chromic acid modification of Bouin’s fluid. However, the peri¬ 
toneum preparations of the osmic fixatives were a little thicker 
and less transparent than the others. If any fixative should be 
exclusively chosen, I believe it should be the chromic acid modi¬ 
fication of Bouin’s solution, because of its excellent fixation, 
convenience, and economy. 

The peritoneum was removed as follows: The two sides of 
the body wall were detached by an incision along the back 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 175 


close to the spine. Under the binocular lens, in water, the 
peritoneum was carefully loosened from the underlying tissue 
by scraping it with a sharp scalpel, first along the edge cut 
from the back. Sections of this loosened edge were then grasped 
by the forceps and relatively large sheets were easily pulled off 
from the underlying tissue. The peritoneum covering the dorsal 
and lateral portion of the body wall is deeply pigmented and to 
it adhere considerable muscle and connective tissue when the 
peritoneum is removed. This portion was grasped with the 
forceps in removing the peritoneum from the body wall, as well 
as in all subsequent handling. Consequently the cells in the 
ventral transparent region available for study have been undis¬ 
turbed by instruments. However, there still remains a possi¬ 
bility that the strain of pulling the peritoneum loose might 
disturb some cells. 

Peritoneum fixed in Flemming’s and Hermann’s solutions was 
stripped from the body wall after four hours of fixation and then 
fixed twenty hours longer. That treated with the various picric 
acid mixtures was stripped immediately after fixation. However, 
the peritoneum fixed in the chromic acid modification of Bouin’s 
solution may be preserved in alcohol for as much as a year before 
stripping. That of Ambystoma punctatum, fixed in Flemming’s 
stronger solution and preserved in 5 per cent formalin, can be 
stripped at least six months after fixation. 

Material fixed in osmic acid fluids was washed five to fourteen 
hours in frequent changes of tap-water. Picric acid preparations 
were gradually transferred to 70 per cent alcohol, beginning with 
10 per cent and progressing through successively stronger grades 
differing by 10 per cent. They remained in each grade five to 
ten minutes. The tissues remained in 70 per cent alcohol con¬ 
taining a few drops of saturated aqueous lithium carbonate 
solution until the picric stain was removed, and before staining 
they were returned to water by reversing the above process. 
All of the material was stained in Heidenhain’s haematoxylin after 
mordanting in a per cent solution of iron alum for four to six 
hours. No counterstains were used. 


JOURNAL OP MORPHOLOGY, VOL. 33, NO. 1 


176 


CHARLES L. PARMENTER 


. Dehydration was accomplished by passing the material through 
the above grades. The fluids were removed from, and added to, 
the containers without handling the material. Alcohols were 
followed by half xylol and half absolute alcohol, and finally by 
xylol. 

The pieces of peritoneum were transferred from xylol to a 
slide where the above-mentioned pigmented area, with the at¬ 
tached muscle fibers, was removed quickly with a sharp scalpel 
just before mounting. After mounting in damar under a cover- 
glass, they were put under a light pressure for twenty-four 
hours or more while drying to insure as flat a preparation as 
possible. 


OBSERVATIONS 

It should be emphasized that the preparations upon which 
these observations were made are unsectioned surface mem¬ 
branes. This makes it possible to study the mitotic figures with 
* the confidence that all of the chromosomes are present and that 
none have been cut and are being counted more than once. 
This is an important consideration in determining whether the 
number of chromosomes is constant. 

A. The number of chromosomes 

There are twenty-eight chromosomes in the somatic complexes 
of Ambystoma tigrinum. In forty-five unquestionable enumera¬ 
tions and in eighteen which contained either one or two chro¬ 
mosomes that might possibly be considered subject to interpre¬ 
tation, there are none which vary from twenty-eight. In three 
complexes, because of the alternative interpretations possible at 
one or more points, the number cannot be definitely determined 
and is interpreted to be either twenty-seven or twenty-eight. 
The fact that these numbers are so close to twenty-eight is 
strong evidence that these cells contain the usual number of 
chromosomes. 

The counts as indicated in the accompanying table have been 
obtained from twenty-three different individuals varying in age 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 177 


approximately from six to ten weeks. The preparations of tail 
epithelium loaned by Professor Sigerfoos were taken from a 
collection which he has been accumulating for a number of years. 
It is probable, therefore, that the counts in these preparations 
represent the chromosome number present during a series of 
years and that the number is constant from year to year. 


Table showing for each tissue studied, the number of different individuals repre¬ 
sented and the number of complexes with their distribution into classes as de¬ 
scribed on page 178. The total number of different individuals represented is 
twenty-three 


TISSUE 

NUMBER 
OP INDI¬ 
VIDUALS 

I 

CLASSES 

II 

ill 

TOTAL 

Peritoneum. 

7 

14 

6 

1 

21 

Mesentery.. 

1 

1 

0 

0 

1 

Lung. 

4 

7 

2 

1 

10 

Tail epithelium. 

5 

9 

4 

0 

13 

Gill plates. 

8 

14 

6 

1 

21 

Totals. 


45 

18 

3 

66 ' 


a. Method of determining number. Since one of the chief pur¬ 
poses of this study is to determine accurately whether there is 
any variation in the number of chromosomes, considerable care 
has been taken to eliminate from the evidence every possible 
source of error. An important part of the presentation of this 
evidence is, then, a concise description of the exact procedure 
employed in obtaining it. 

1. Procedure. In order to avoid overlooking any mitotic 
figures, the entire surface of every piece of tissue was completely 
surveyed systematically before beginning to count any of the 
chromosomes in any of the complexes. The survey was accom¬ 
plished with a 4-mm. objective and an 8X ocular supplemented 
by a mechanical stage. 

In determining the number of chromosomes in each complex, 
a camera lucida sketch of it was first made at a magnification of 
2633 diameters. This sketch was carefully compared with the 
cell in order to make certain that no errors had been made in 




















178 


CHARLES L. PARMENTER 


sketching it. The chromosomes were then numbered consecu¬ 
tively, the number being placed on both ends of each chromo¬ 
some. This method avoided any possibility of overlooking any 
chromosome or of counting any chromosome twice. 

2. Clearness and classification of the complexes. All the com¬ 
plexes counted were polar views of late prophases and of meta¬ 
phases and have been divided into three classes on the basis of 
their clearness. The first class consists of forty-five complexes 
in which every chromosome was so clearly separated from adja¬ 
cent chromosomes that it could be optically traced continuously 
over its entire length, without losing sight of it at any point. 
Only the counts from complexes of this group are submitted 
as data which are unquestionably free from objection and 
uncertainty. 

In the second class of cells there are eighteen complexes in 
which the chromosomes are all exactly as clear as those of the 
first class, with the exception that either one or two chromosomes 
cannot be clearly traced over their entire length as they could 
be in class I and therefore might possibly be hypercritically 
considered to necessitate interpretation. 

The three cells of the third class differ from those of the 
second class in that they each contain places in which the number 
of chromosomes cannot be determined with confidence and 
consequently are actually subjects for interpretation. 

Complexes of the first class. Complexes of this class are 
represented by figures 1 to 8 which have been made in carbon 
and are attempts to represent the actual appearance of the 
chromosomes and their relative positions in the complexes. Rep¬ 
resentative cells from each of the tissues studied, except the 
mesentery and lung, have been so drawn. Other complexes of 
this group have been outlined in ink, figures 9 to 20, to give a 
further assurance of the nature of the complexes constituting 
this class of conditions. 

Since it is impossible to represent chromosomes in a drawing 
as clearly as they are seen in a cell, it is necessary to consider 
briefly this situation in order to prevent misunderstanding, and 
incorrect impressions concerning the clearness of the cells and 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 179 


the faithfulness of the description of the conditions under which 
the number of chromosomes was determined. The difficulty 
lies in the necessity of representing on a plane surface chromo¬ 
somes which in the cell occupy several levels. The effect can 
be produced by shading, but at the same time at points where 
chromosomes cross or overlap each other for various distances 
they might create the impression in the drawing that they 
cannot be “ optically traced continuously over their entire 
length.” There are such cases in every drawing. This is 
especially true of the late metaphases of the tail epithelial com¬ 
plexes (e.g., figs. 7, 8) where every chromosome in the cell can 
be clearly and faithfully traced as described above. 

There is also the condition in which parts of the same chromo¬ 
some are so related to one another that their appearance in the 
drawings might create a doubt as to their clearness in the cell. 
Examples of this are represented in figures 6 and 8, chromosome 
‘ a/ in which the two arms of the same chromosome turn abruptly 
upon one another and the appearance might be subject to the 
criticism that there are two different chromosomes involved—a 
portion of one lying exactly upon another with their ends termi¬ 
nating at the same point. Such cases were carefully examined 
end the two arms can clearly be seen to follow into each other. 

In four of this first class of cells there is another condition that 
needs mention. These cells contain one or two chromosomes 
which appear to be broken into two parts (e.g., figs. 19 and 20, /). 
The parts in each case are separated by very short spaces and 
are exactly in line with each other. Della Valle (’09, fig. 11) 
shows two cases of this sort as one chromosome, but discusses 
them (p. 116) as uncertain. That there is a single chromosome 
concerned in each of these cases is further evidenced by the fact 
that there are twenty-one similar cases in other cells of this 
class (e.g., figs. 5, 7, 14 and 15, /) and thirty-five cases in cells 
of class II in which the parts are connected by various amounts 
of chromatin. In some instances the connection is seen as 
faintly staining chromatin, in others as a single or double darkly 
stained thread. 


180 


CHARLES L. PARMENTER 


Complexes of the second class. In fourteen cells of this class 
there is one point in one chromosome and in four cells there is 
one point in each of two chromosomes which, to persons hyper- 
critically inclined, might possibly appear uncertain. To one 
acquainted with the material, each of these points is entirely 
clear, and even when accepted as subject to interpretation it is 
very plain how the interpretation should be made—so plain that 
I am certain that the count of twenty-eight chromosomes is 
accurate and dependable. But for the sake of unquestionable 
fairness I have placed these cells in a separate group. As to 
the exact nature of the interpretations in these eighteen com¬ 
plexes, four of them have some small portion of only one chro¬ 
mosome so covered by others that it cannot be traced over its 
entire length without losing sight of it as stated above (p. 178). 
Two other cells had two chromosomes of this nature. Five 
complexes have a single chromosome lying in such a relation to 
another chromosome that it might possibly be interpreted as a 
part of the other chromosome (e.g., fig. 23, chropiosome i), and 
in three more cells there were two such chromosomes. In the 
remaining five complexes a single chromosome was so situated 
or otherwise involved, that it might be interpreted that there 
were two chromosomes present (e.g., fig. 21, i). 

In considering all the interpretation possible in each of these 
eighteen cells the minimum number in any one of them would 
be twenty-seven and the maximum number thirty. Even grant¬ 
ing this much variation, it is far removed from that expected in 
a series of chance variants as Della Valle claims them to be. 

The points in question were sketched as described above 
before the chromosomes were counted, so that the determination 
of the number of chromosomes was not influenced, either con¬ 
sciously or unconsciously, by a knowledge of how many chromo¬ 
somes were present or by how they should be sketched in order 
to produce the expected number. This procedure and the fact 
that the number counted always agreed with the number present 
in the forty-five cells of class I make it practically certain that 
the enumeration is correct. It should be emphasized again that 
these cases are only subject to question when hypercritically 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 181 


considered and would otherwise constitute a part of class I. In 
fact, an experienced cytologist of this laboratory, in examining 
these, without a knowledge of the number of chromosomes 
present, could see no reason for considering them as subjects 
for interpretation, and it seems almost absurd to place them in 
a separate class. 

Complexes of the third class. There was a very large number 
of cells which were beautifully clear everywhere except in regard 
to one or two chromosomes. However, only three of these were 
sketched, because the number of clear counts was so large that an 
increased number of these uncertain counts is of little value. 

Each of the three cells drawn contains two points of uncer¬ 
tainty as to whether there are one or two chromosomes present. 
The number is interpreted as either twenty-seven or twenty-eight. 
The minimum number of chromosomes possible of interpreta¬ 
tion in one cell is twenty-six, the maximum is twenty-eight; in 
the other two cells the minimum is twenty-seven, the maximum 
is twenty-nine. 

These three cases were interpreted while the sketch was being 
made and before it was known how many chromosomes were 
present. It is not true, therefore, that the interpretations were 
prejudiced nor that any cases which did not agree with the 
expected numbers were cast aside and consequently ignored. 
On the contrary, they are here included as part of the evidence in 
forming the conclusions drawn from this study. 

Rationally considered, then, of the cells sketched there are 
sixty-three in which the enumeration of chromosomes is accurate 
and dependable and three in which there are unavoidable 
interpretations necessary. These sixty-six complexes constitute 
very strong evidence that the number of chromosomes in Am- 
bystoma tigrinum is constant. 

b. Possible variation in number in uncounted complexes. As to 
whether or not there was any variation in chromosome number 
in this species can be judged from the results obtained from the 
sixty-six cells which were studied. If as few as 2 per cent of the 
total complexes studied varied from the usual number, at least 
one of these should have made its appearance. Furthermore, 


182 


CHARLES L. PARMENTER 


Della Valle (’09, p. 117)' claims that variation of chromosome 
number is probably a general law and (p. 120) that his counts 
strikingly bear out the expectation expressed by Newton’s theo¬ 
retical binomial curve. Were this the condition in Ambystoma 
tigrinum, a good proportion of the sixty-six complexes should 
have shown variation in number. Since no variation was found, 
it is safe to conclude that there is none in the cells that could 
not be counted. 

c. Abnormal complexes. Seven apparent variations from the 
usual number were found. These were groups of chromosomes 
in which the number was clearly other than twenty-eight (figs. 
22, 24, 25, A.B., 26, A.B.). But when these groups are thoroughly 
analyzed it is certain that they are nothing else than cases of a 
very unusual behavior of four cells and do not constitute a 
variation from the usual number of chromosomes. 

Figure 22 shows a peritoneal cell which has lost a part of the 
chromosomes. Chromosome a is but part of a chromosome, 
showing very unmistakable evidence that a portion of it has been 
broken off and there is a conspicuous depression in the tissue 
from which it is evident that the remainder of the chromosomes 
of this cell have been lost. The cell lies close to a tear in the 
peritoneum. It is a bare possibility that the tear and the loss 
of the chromosomes is due to the same cause. 

The second case is a very early metaphase from the peritoneum. 
It consists, as represented in figure 24, of one group of twelve 
chromosomes and another group of sixteen immediately adja¬ 
cent to it. These two groups and figures 2 and 20 are very 
similar to Della Valle’s dicentric cell (’09, fig. 6) and Flemming’s 
(’91) figures 31 to 39, table 40. A study of these two groups 
makes it practically certain that they are separated parts of one 
and the same cell. This is evidenced by the following facts: 1) 
these two groups together constitute the normal number twenty- 
eight. 2) The chromosomes of both groups of cells are in the 
same stage of mitosis. 3) Both groups represent a half circle 
and indicate strongly that they are separated parts of one cell 
wdiich have rotated a total of 180° to their present positions. 4) 
When these chromosomes are arranged side by side linearly they 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 183 


form a series (fig. 31) like that (figs. 27 to 30) made by a similar 
arrangement of the chromosomes of normal cells (figures 1 and 
3). The length relationships of these chromosomes as shown 
graphically in figure 37 are practically identical with those of 
normal cells (figs. 33 to 36). Unfortunately, the cell walls are 
not visible. 5) Both of the homologues of chromosome pairs, 
as determined by measurements and indicated in figure 24 by a 
duplicate series of numbers, in some cases are found in the 
same separated part of the cell and in other cases one homologue 
is found in part a and the other in part b. 

The third case is a compact metaphase in the epithelium of 
the lung (figs. 26 A and B) and is similar to the second case. 
These two groups are somewhat more separated than those of 
figure 24. Figure 26 B represents the chromosome number and 
characteristics and figure 26 A shows the relative positions of 
the two groups omitting some of the chromosomes in a. 

That these two groups of chromosomes are parts of the same 
cell which have become separated is made highly probable by 
the following facts: 1) As in the second case (fig. 24), the two 
groups are near together, one containing eight and the other 
twenty chromosomes—a total of twenty-eight. 2) The chromo¬ 
somes are in the same stage of mitosis, the chromatids of those 
of b, however, being separated a little more than those of the 
twenty chromosomes in a which may be due to a less crowded 
condition. 3) The two groups are practically of the same 
diameter and of the same shape. An outline of a on transparent 
paper can be perfectly fitted to b. 4) These chromosomes also 
form a linear series of lengths (fig. 32) similar to those of normal 
cells. 5) Group a is not a complete cell because the cytoplasm 
can be seen only below the chromosomes, while above the 
chromosomes are bare. The boundaries of the cytoplasm of b 
cannot be seen. 6) The homologues of the chromosome pairs 
are numbered and distributed in the two groups like'those of 
figure 24. 

The fourth case is the peritoneum of another individual. It 
evidently is a cell which has been divided into two parts like 
those of cases 2 and 3. Figure 25 A is a camera-lucida drawing 


184 


CHARLES L. PARMENTER 


representing the relative positions of the two groups and figure 
25 B shows the chromosomes enlarged and numbered consecu¬ 
tively. As in case 3, the two groups are not immediately 
adjacent, but are separated by the longer diameter of a resting 
nucleus. 

In group a there are eleven chromosomes. In the other group 
there are apparently seventeen, but unfortunately in this second 
group the chromosomes are so overlapped at one point that 
they cannot be counted with confidence. There are, however, 
thirteen chromosomes which can be clearly delineated and the 
interpretation that there are four chromosomes in the group 
(14 to 17) which so badly overlap is likely correct. The total 
number of chromosomes in the two groups is then probably 
twenty-eight. 

The chromosomes are so much foreshortened, and at the 
above-mentioned point so crowded, that I have not attempted to 
measure and arrange them in a series as was done for the chro¬ 
mosomes of figures 24 and 26. However, a glance at figure 25 B 
shows that such a series might be arranged. 

The shape and size of both groups of chromosomes, and of 
the cytoplasm about them, are such that one can be fitted upon 
the other. Although these two relations are not positive evidence 
they indicate that one of these groups, possibly the smaller, has 
been separated from the other. 

To summarize, it may be said that in the first case considered 
(fig. 22) it is certain that the smaller number of chromosomes is 
due to a loss of a part of the chromosome complex from the cell. 
Although the facts stated for cases 2, 3, and 4 may not be con¬ 
sidered absolute proof, they do constitute a very strong prob¬ 
ability, which closely approximates a proof, that in each of these 
cases a cell has been separated into two parts. 

B, Somatic chromosome pairs 

a. Introductory statement. Since Van Beneden’s (’83) hypoth¬ 
esis that one-half of the chromosomes of an individual are of 
paternal origin and that one-half are of maternal origin there 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 185 


have been many confirmatory observations and some that 
oppose it. 

Montgomery (’01, p. 220) advanced evidence that for each 
of the chromosomes of maternal origin there is a homologous 
mate among the chromosomes of paternal origin, and that these 
homologues unite during synapsis. He also maintained that 
these pairs 3 can be recognized in the spermatogonia. This 
view has been supported by many authors. Among these are 
Sutton (’02), who compared numerous camera-lucida drawings 
of spermatogonial complexes of Brachystola magna; Meek 
(’12 a), who measured the lengths of spermatogonial chromo¬ 
somes of a somewhat wide range of animals, and Hance (’17 b, 
’18 a), who made linear measurements on the germinal and 
somatic chromosomes of the primrose, Oenothera scintillans and 
the pig. On the other hand, Meves (’ll), on the basis of meas¬ 
urements made upon the spermatogonial and somatic complexes 
of Salamandra maculosa, fails to confirm the claim for the 
former and denies it (p. 282) for the latter. Della Valle (’12), 
who measured the chromosomes of peritoneal cells of the same 
form, also denies the existence of pairs. 

Some of the somatic cells studied in Ambystoma tigrinum are 
quite favorable for a linear measurement of chromosomes, and 
these complexes have been used to obtain further data upon 
the query as to whether the chromosomes of the somatic cells 
form a duplicate series (based upon length and form) as is shown 
by their progenitors in the germinal line during the maturation 
period. 

b. Mensuration. Since the possibilities of error in measure¬ 
ments are so great, it is necessary to consider the conditions 


8 The two mates constituting a pair are usually of equal length, so that homo¬ 
logues may be recognized by such equality. In some cases, for example, in 
the Diptera, Stevens (’08, ’ll), Metz (T4, T6 a and b), Holt (’17), Whiting (’17), 
Hance (’17), the two members lie near each other or even closely approximated 
in the spermatogonia and somatic cells, while in many other cases, for example, 
in Orthoptera and Amphibia, the homologues may be widely separated in these 
cells. In the present paper the term ‘pairs’ will refer to the two chromosomes 
which are homologues as determined by length and form regardless of the 
relative position in the cell. 


186 


CHARLES L. PARMENTER 


under which these measurements were made in order to judge 
their value correctly. 

1. Type of cells. Only cells were used in which every chro¬ 
mosome was perfectly clear and, except as noted (p. 189), lay 
exactly level in the equatorial plane throughout their entire 
length. Only three cells (figs. 1, 3, and 9) of this quality 
were available, and these were polar views of early metaphase 
stages in cells of the peritoneum and lung. The chromosomes 
of one other cell (fig. 10) approximated this condition and were 
also measured. The care with which these cells have been 
chosen may be judged from the fact that they were the only 
suitable cells in material from over one hundred larvae con¬ 
taining large numbers of division figures. In material with chro¬ 
mosomes so long and so numerous it is not surprising that so few 
cells were perfect enough for measurement. 

2. Method. In addition to choosing cells with chromosomes 
of the above character, three different camera-lucida sketches of 
each chromosome were made on different days with extreme care 
at a magnification of 2633 diameters. Each of these sketches 
was measured three or more times along the median line with an 
Ott compensating planimeter modified for this purpose, or with 
an opisometer. These nine determinations obtained for each 
chromosome were averaged to represent its length. This method 
is important because the extremes of these nine measurements in 
about one-fifth of the cases may differ 1 mm. from the average 
(and occasionally more). This demonstrates that one measure¬ 
ment upon a single drawing might give rise to an erroneous 
difference in the lengths of the homologues of some pairs rang¬ 
ing from 1 to 2 mm., the actual amount depending upon the 
respective errors in each homologue. Averages largely eliminate 
this error. 

3. Sources of error. The various sources of error may be classi¬ 
fied in three groups: 1) instrumental errors, 2) personal errors, 3) 
errors inherent in the condition of the material. 

In the first place, it should be emphasized that no attempt 
has been made to determine the actual length of any chromo¬ 
some. These measurements have all been made on the drawings 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 187 


described above and have to do only with relative lengths. This 
fact eliminates at once a number of errors which would otherwise 
be very serious. 

The instrumental errors. The possible instrumental errors are 
a) failure to maintain a critical illumination, b) failure to maintain 
a constant wave length of illumination, and c) errors inherent in 
the planimeter and opisometer. 

It so happened that the strongest light was obtained a little 
above the point of critical illumination, and might therefore 
cause an error in measurement. However, several chromosomes 
were drawn a number of times under both conditions and no 
perceptible difference was observed. Any slight error overlooked 
would be equal in the homologous chromosomes and would not 
interfere with a relative measurement. 

Farmer and Digby (T4) state that errors can arise from the 
use of varying wave lengths of light. The same optical equip¬ 
ment and illumination were maintained in all of my operations 
so that relative values were unaffected. 

In making measurements with the planimeter the polar arm 
was held rigidly stationary in two grooved blocks so that the 
tracing point moved around in a circle having a diameter of 33 
cm. The sharp tracing point was kept upon the median line of 
the chromosome figure by moving the drawing around (without 
slipping) into line with the path of this tracing point, which was 
used as a pivot for orienting the drawing. A constant, repre¬ 
senting the value of each of the divisions of the vernier, was 
determined by measuring a series of known distances on a straight 
line. 

The accuracy with which this instrument was operated is 
indicated by the fact that the average difference between the 
extremes of measurements made upon each of several drawings 
is 0.3 mm., the standard deviation, computed from the combined 
measurements ,of several drawings, is 0.17 mm. The measure¬ 
ments, obtained more quickly with the opisometer, are slightly 
less accurate, the average of the above extremes and the standard 
deviations being 0.4 mm. and 0.26 mm., respectively. Finally, 
the average of the nine measurements made upon the three 
drawings of each chromosome reduces this instrumental error 


188 


CHARLES L. PARMENTER 


to approximately zero. This error is, of course, inherently 
included as a part of the personal error discussed below. 

Personal errors. Probably the greatest personal error was 
due to inaccuracies in making camera-lucida drawings. To 
reduce this error to a minimum, each chromosome, as stated 
above (p. 186), was drawn three times with extreme care. These 
sketches were made at the same point on the drawing-board so 
that any error due to different drawing distances and consequent 
differences in magnification was eliminated. The estimated 
median line of the sketch, upon which the measurement was 
made, was indicated with a lead pencil. The average deviation 
from the mean of the nine measurements is 0.6 mm. and the 
standard deviation, computed from combined measurements of 
several drawings, is 0.37 mm., which indicates that the instru¬ 
mental and personal errors in the average of these measurements 
are practically zero for relative purposes. 

Errors due to conditions inherent in the material. This class 
of errors is much more important than the preceding. The 
errors of this kind are an unequal shortening of the whole chro¬ 
mosome and a foreshortening of parts or all of the chromosome. 
Measurements made without very careful attention to fore¬ 
shortening are of questionable value, for small amounts can 
give rise to large errors, especially in short chromosomes. Shorten¬ 
ing is caused by a twisting of the chromatids about one another 
(figs. 1 to 8, 27 to 30). The amount of shortening in each twist 
of the chromatids, as determined by computation, 4 at the mag- 

4 The amount of shortening in each twist of a chromosome was determined 
by adding together the separately computed amounts of shortening due to the 
lateral deviation of the chromatids and that due to the vertical deviation of 
the chromatids. The shortening in each twist due to the lateral deviation was 
computed by averaging the lengths of the two chromatids of a chromosome and 
substracting the length measured upon the median line of the whole chromo¬ 
some. This total difference divided by the number of twists is 0.2 mm., which 
is approximately the amount of shortening due to the lateral deviation in each 
twist. In determining the amount of shortening due to the vertical deviation 
of the chromatids, the width or thickness of the chromatid, as determined with 
an ocular micrometer was assumed to be the amount of vertical sag of the chro¬ 
matid in each twist. ' This thickness multiplied by the magnification amounted 
to 1 mm. This was used as the altitude of a right triangle, the base of which 
represented half of the measured longitudinal length of the shortened part, and 
the hypotenuse of which then represents very closely one-half of the true length 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 189 


nification of the drawings (2633 diameters), amounts very 
closely to 0.4 mm. However, the effect of this condition is 
either completely or largely neutralized by equal or nearly equal 
amounts of twisting in the homologues of each pair. The maxi¬ 
mum amount of error due to this cause may be judged by an 
examination of figure 28 which contains the most twisting. In 
this cell there are seven pairs in which the amount of twisting 
is equal and the error completely neutralized, two pairs con¬ 
taining an error of 0.4 mm., two pairs with 0.8 mm. error, one 
with 1.2 mm., and two pairs in which it is uncertain. Since 
these errors that occur at critical points will be considered indi¬ 
vidually later, no corrections for them are included in the 
measurements. 

Foreshortening occurs only in certain chromosomes as indi¬ 
cated in figures 27 to 30 and 33 to 36. This is, however, in the 
cells represented by figures 27, 28, 33, and 34 so slight that the 
whole chropiosome can be seen at one focus, the foreshortened 
part appearing only slightly hazy. In the cells represented by 
figures 29, 30, 35, and 36 it is a little more. Measurements 
with the fine-adjustment graduated wheel, made more accurate 
with a sharper pointer made of a pin, indicated this sagging to 
be not more than 2.5/x (one division of the fine adjustment wheel) 
in any case. Corrections 5 made for this foreshortening are 

of the shortened portion. Double the length of this hypotenuse minus the orignal 
measurement of the shortened portion is 0.2 mm., which is the maximum amount 
of shortening due to the vertical sag of any twisted portion. This amount added 
to that caused by the lateral deviation made the total shortening in one twist 
amount to 0.4 mm. Although this determination cannot be considered entirely 
accurate, it is a close approximation. 

6 The correction was made as follows. The amount of vertical deviation was 
read from the fine-adjustment wheel when the objective was focused as nearly 
as could be judged upon the middle of the lowest and highest points of the fore¬ 
shortened portions. All measurements for a given complex were made with the 
same part of the fine-adjustment screw, thus avoiding different pitches in the 
thread. The reading (2.5 n for each division) gave the actual differences of 
vertical positions. This multiplied by the magnification and divided by 1000 
converted the figure into millimeters, the units made use of in the drawings. 
By using this distance as the altitude of a right triangle and the measured longi¬ 
tudinal extent of the foreshortened portion as the base of the triangle, the hypot¬ 
enuse (which represented approximately the correct length of the foreshortened 
part) was determined. This was substituted for the original measurement of 
the foreshortened portion. 


190 


CHARLES L. PARMENTER 


only approximate, because it is very difficult to determine 
accurately the amount of vertical deviation, and its longitudinal 
extent, as well as its exact course. In figures 27 to 30 and 33 
to 36 corrected figures are used, and the amount included in each 
measurement for foreshortening is indicated. 

There are two other conditions which do not give rise to actual 
errors in measurement, but do interfere with precision of results 
and may well be considered here. 1) A possible unequal con¬ 
traction of chromosomes. Wenrich (T6) observes that chromo¬ 
somes A and B condense before the other chromosomes in the 
spermatogonia and tetrads of Phrynotettix, and (T7) he shows 
that one homologue of chromosome 4, cell E, plate 2, contracts 
more rapidly than the other. 2) Since so many of the chromo¬ 
somes of Ambystoma are so long and composed of two inter¬ 
twined chromatids, there is considerable possibility of a stretching 
due to bending and other stresses still present in the complexes 
nearing the metaphase. As Meves ('ll, p. 247) points out, 
under these conditions two chromosomes could be of different 
length and of equal volume. Even an imperceptible difference 
in diameter of parts or all of two chromosomes of equal volume 
might cause considerable difference in their lengths. This dif¬ 
ference would of course be proportionally greater in the longer 
chromosomes so that measurements of the shorter chromosomes 
of a cell might strongly indicate the presence of pairs while the 
homologues of the longer pairs would show quite wide differences 
in length. A case of very perceptible stretching is to be seen in 
chomosomes ‘s,’ figures 9 and 12. 

Effects of technique. The differences which may arise in the 
chromosomes of different cells of even the same tissue due to 
different effects of fixatives, and all other effects of technique, 
do not affect relative measurements of chromosomes in the same 
cell. For it is extremely improbable that the lengths of chro¬ 
mosomes of the same cell which are so equally close to the 
surface of these membranes would not be similarly affected by 
the action of these various reagents and processes. It is also 
improbable that inherent differences among the homologues 
would cause a differential change of length under these 
conditions. 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 191 


Summary. The combined instrumental and personal errors 
are reduced to practically zero by averaging several measurements 
made upon different drawings. The errors due to twisting of 
chromatids about one another are largely neutralized and are 
considered individually later. Therefore, except for possible 
unequal contraction and stretching of homologues, only the 
measurements of those chromosomes which are foreshortened 
contain appreciable errors. It is thought that these errors, 
after corrections have been made, probably do not in any case 
exceed 1 mm. The presence and amount of error due to unequal 
contraction and stretching in any particular chromosome is an 
uncertainty, but if existing would probably be greater in the 
longer chromosomes. 

c. Results of measurements. 1. Criteria for determining pairs. 
Before considering the results of the measurements, it seems 
desirable to state what the criteria are that will demonstrate 
whether pairs (p. 185) are present among the chromosomes at 
these particular stages of mitosis. In the absence of definite 
minute morphological characteristics, such as a repeated occur¬ 
rence of marked granules, constant in position and size, which 
Wenrich (’16) describes for certain Orthopteran chromosomes, 
the next most exact criterion for determining the presence of 
pairs in diploid cells would be a duplicate series of chromosomes 
of equal volume. But in these chromosomes trustworthy volu¬ 
metric determinations cannot be obtained, for the above- 
mentioned intertwining of the chromatids and the stretching of 
the chromosomes would cause variations in diameter which 
could not be measured accurately, and these errors would be 
cubed in the volume. Consequently linear measurements, sup¬ 
ported by form, have been chosen as giving more trustworthy 
data. 

Upon this basis, in order to constitute undeniable evidence 
that the chromosomes form a duplex series, there are two con¬ 
ditions which should be met. First, when the chromosome 
lengths are plotted in a graph (e.g., figs. 33 to 37), they should 
definitely associate themselves in twos of equal lengths. Second, 
the differences in length between successive pairs, as indicated 


JOURNAL, OF MORPHOLOGY, VOL. 33, NO. 1 


192 


CHARLES L. PARMENTER 


by the first condition, should exceed the errors of measurement 
by a good margin. Unless the above conditions are met, the 
errors of measurement make it possible to contend that the 
chromosomes are arranged in a series of successively increasing 
lengths which bear no relation to one another and therefore do 
not represent pairs. 

In addition to the above, the form of the chromosome, which 
is probably determined in large part by the position of the 
spindle fiber attachment, may be used as an aid in determining 
which chromosomes are homologues. McClung (’14, p. 674) 
pointed out that, although the spindle fiber attachment may be 
different on different chromosomes, nevertheless, for each chro¬ 
mosome “it is most precise and constant” in the individual. 
Carothers (’17, p. 470) has shown that for certain tetrads (e.g., 
figs. 32 and 63) the point of spindle fiber attachment on one 
homologue is different from that on the other. But she also 
finds that the point of fiber attachment is constant on a given 
homologue for each individual. She shows (figs. 32, 32 a and 
63, 63 a) that the point of spindle fiber attachment on these 
homologues in the spermatogonia is preserved in the tetrads. 
However, an exception to constancy of fiber attachment in the 
individual has been noted by Wenrich (’16). He found in a 
rod-shaped tetrad of another genus that the fiber attachment 
might shift from one end of the chromosome to the other in 
certain individuals. Therefore, according to the theory of the 
individuality, in the somatic chromosomes the homologues of 
certain pairs of chromosomes may be expected to be unlike in 
form. However, individuals showing such conditions are very 
few and should be considered exceptions rather than the rule. 
There is the possibility that these heteromorphic homologues 
may not be confined to the Orthoptera, and certain cases in 
Ambystoma make this appear to be so. 

Finally, it would be remarkable if any material satisfied the 
above criterion in all points. The small difference in length 
between some chromosomes makes it impossible to demonstrate 
beyond doubt the presence of pairs among them. Again, the 
possibility that the chromosomes may not condense at equal 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 193 


rates, and that unequal stretching, especially between the 
longer chromosomes, may occur during mitosis, increases the 
difficulty in obtaining accurate metric comparisons, and may 
interfere with perfect certainty in the interpretation of the 
results. Furthermore, some variation is characteristic of living 
material and hence slight differences in relative length and form 
in different cells would be expected rather than absolute uni¬ 
formity. McClung (’17, p. 567) finds in certain Orthoptera 
that the accessory chromosome, although unmistakably dis¬ 
tinguished from the euchromosomes, is not always of the same 
relative length in different individuals of a given species, for it 
sometimes occupies the fourth and sometimes the fifth position 
in the series of lengths. However, it must be remembered that 
this is the sex chromosome which at the metaphase (the stage 
of greatest condensation of the euchromosomes) is already be¬ 
coming diffuse. Differences in the degree of condensation might 
therefore be involved in the differences of relative lengths. And 
again, since individuals vary in their morphological character¬ 
istics, why should it be expected that the chromosomes of two 
different individuals should be of exactly the same relative 
lengths at the same stage of mitosis? In view of universal 
variability, homologous chromosomes, which are derived from 
different individuals and which may be expected to maintain 
their individuality, should not invariably be of exactly the 
same length. As discussed (p. 217), the observations on different 
Orthoptera by several authors indicates this to be so. 

On account of these interfering factors it cannot be expected 
that homologous chromosomes will always be of exactly the 
same length at any particular stage of mitosis. Therefore, 
length and form, considered in a limited number of cells, from 
different individuals, cannot be regarded as conclusive evidence 
for or against the presence of chromosome pairs. Much more 
conclusive evidence would be had in a comparison of several 
somatic complexes from a single individual and with those of 
other individuals, a comparison of these with the diploid and 
haploid chromosomes of the germinal line, and a comparison of 
the complexes of parents and progeny. 


194 


CHARLES L. PARMENTER 


However, although the measurements may not meet the above 
criteria in all chromosomes, there are certain cases which do meet 
them definitely, and strongly evidence the existence of pairs. 
This fact, together with the above consideration, makes it 
possible that all the chromosomes are in pairs. 

Furthermore, it may be mentioned here that conditions which 
do not meet the above criteria fall far short of proving that 
pairs do not exist. The possibility still remains that two or 
more pairs may be of equal or nearly equal length. Such a 
condition is known to exist in certain Orthopteran chromosome 
pairs (Carothers, T7, pi. 1, tetrads 7 and 8) where the chromosomes 
are unquestionably known to be paired. 

2. Evidence for the existence of pairs. On plate 9, figures 33 
to 37, are five rows of vertical lines representing the relative 
lengths of the chromosomes of as many cells. The differences 
in the lengths of these lines and also the space between adjacent 
lines represent relative differences in chromosome length. For 
convenience the lines are made twice the length of the chromo¬ 
somes as drawn and the width of the spaces are made eight times 
the difference in length. The lengths of the chromosomes, the 
amounts included for foreshortening, and the form of chromo¬ 
somes for each of these cells are also shown, respectively, in 
figures 27 to 31 and 33 to 37. 

A part of the evidence which these graphs present for the 
existence of pairs is three outstanding characteristics common 
to all of them. First, there is a graded series of chromosome 
lengths from the shortest to the longest; second, there is a 
marked sameness in the relative chromosome lengths of these 
cells which appears in the approximately constant presence of 
groups containing the same chromosome pairs, and, third, a 
similarity of form between homologues. 

The pairs, in accordance with the above criteria, were de¬ 
termined primarily on the basis of chromosome length, supported 
by a comparison of form. The graphs and figures of each com¬ 
plex measured show that certain chromosomes are very probably 
homologues. In other cases a number of chromosomes are so 
nearly of the same length that, according to the criteria, the 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 1.95 

homologues of the pairs cannot be determined with certainty, 
but they doubtless exist. Since the measurements of figures 33 
and 34 are very nearly accurate and present the most reliable 
evidence, these will be considered separately from the others. 
For convenience the pairs may be considered in two groups, the 
first including those which differ greatly in length from their 
neighbors (pairs 1, 2, and 8) and the second including the 
remainder in which the pairs are not so clearly distinguished. 

It is all but certain that the chromosomes represented in each 
of pairs 1, 2, and 8 in both of these figures are homologues, for 
they almost completely satisfy the criteria outlined above. In 
these pairs there is foreshortening in only one chromosome and 
in pair 8 the error of 0.8 mm. in both complexes due to twisting 
of the chromatids is negligible. The homologues are of approxi¬ 
mately equal length, and the difference between each pair and 
the adjacent pairs is so much greater than the error of measure¬ 
ment that it is improbable that the condition represented by 
these three pairs in both complexes is merely a matter of chance. 
Furthermore, there is a close resemblance of form between these 
homologues. A comparison of other cells of the same individual, 
if available, would be expected to show that this condition is 
constant in all the cells as is shown by comparable cases of 
constancy in the germ cells of individuals of certain Orthoptera 
(p. 219). It can, therefore, be maintained with considerable 
confidence that these particular chromosomes of equal length 
and sameness of form actually constitute pairs. The measure¬ 
ments of the chromosomes of these pairs in other cells as dis¬ 
cussed below give similar although less conclusive evidence. 

Among the chromosomes of the second group in these two cells 
there is strong evidence for the existence of pairs, but the small 
difference in length and the errors due to twisting and possible 
stretching make it inconclusive. In figure 33 the homologues as 
shown in each of pairs 3 to 7 are so nearly of the same length 
and form (fig. 27) that one may believe that they constitute 
pairs as represented. Pair 3, in addition, is well separated from 
those adjacent. Although pairs 4 to 7 appear to be actual 
pairs, the chromosomes of this series differ so little in length that 


196 


CHARLES L. PARMENTER 


the criteria adopted are not entirely satisfied. There is a chance 
of doubt, therefore, of the validity of the pairs as indicated. 

As mentioned above, it will be noted that the groups into 
which the chromosomes of this cell are associated are repeated 
in the other cells. The similarity of grouping is very marked, 
especially in the formation of two large and distinctly separated 
groups, one containing pairs 3 to 7 and the other pairs 9 to 14. 
In pairs 3 to 7 of figure 34 the condition present in figure 33 is 
duplicated, except that pair 3 is not so well separated from pair 
4, due to the fact that both homologues of pair 4 are relatively 
shorter in the former complex. Concerning the homologues of 
pair 6 there is some doubt. I have interpreted the end of 
chromosome 30 + (fig. 28) as bending back upon the main 
portion of the chromosome, and have estimated the length of 
this portion. 

Of the remaining six pairs (9 to 14) in both cells several are 
quite clear, but on the whole the possibilities of error and the 
differences in length between successive pairs is too small to 
satisfy the second criterion fully. In figure 33, on account of 
the practical absence of twisting in pair 12 and adjacent pairs, 
the condition for determining pairs is very closely satisfied. 
The chromosomes of pair 9 were considered homologues through 
a process of elimination. They differ 12.4 mm. in length, but 
agree in form (fig. 27). This condition will be discussed later 
(p. 217). 

In figure 34, pairs 9, 11, and 13, although not sufficiently 
separated to constitute an unquestionable demonstration of pairs, 
are fairly well separated and the homologues of each pair, after 
allowance is made for errors due to twisting, are of nearly equal 
length. One homologue of pair 10 (fig. 28) is imperfect. Pair 
14 is well separated in the graph from pair 13. Approximate 
corrections made for the kink in the shorter member and for 
the slight foreshortening at that point make its length approxi¬ 
mately the same as that of the longer member. The homologues 
of all these pairs agree very well in form (fig. 28), in spite of the 
fact that some may not yet have assumed their final shape. 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 


197 


The measurements represented in figure 35 are nearly as 
reliable as those of the preceding cells. The amounts included 
for very slight foreshortenings are indicated in figure 29. The 
relative chromosome lengths form approximately the same 
groups as those of the other cells, and the evidence for the 
existence of pairs is strong. As contrasted with figures 33 and 34, 
the chromosomes of pairs 1 and 2, when approximately corrected 
for foreshortening, do not entirely meet the conditions of the 
criteria, but their lengths and form strongly support the prob¬ 
ability that they are homologues. The chromosomes of pairs 7, 
8, and 10 differ somewhat in length. They do not appear fore¬ 
shortened, and although possibly present there is no perceptible 
stretching in them. These differences may be due to an un¬ 
equalness of homologues as discussed later (p. 217). On the 
other hand, the chromosomes of pairs 3 to 6, 9 and 11 to 13 are, 
except as noted in figure 29, unforeshortened and of equal length, 
they agree in form and present strong evidence for the exist¬ 
ence of pairs. The greater part of the difference in length 
between the homologues of pair 14 is due to stretching. 

The measurement of the chromosomes of the cell represented 
in figures 30 and 36 are somewhat less favorable for measurement 
than those of the preceding cells, because in addition to a 
moderate amount of twisting there is slight foreshortening in 
many of them. Although the lengths have been approximately 
corrected for this foreshortening (figs. 30 and 36) the measure¬ 
ments cannot be considered so accurate and reliable as those of 
figures 33 and 34. The relative lengths of the pairs closely 
parallels that of the preceding figures which results in a similar 
distribution in the series. As contrasted with figures 33 and 34, 
the chromosomes of pairs 1 and 2 fail to satisfy the criteria, 
and this is apparently not due to errors in measurement. The 
large difference of 5.6 mm. between the homologues of pair 7 
recalls a similar difference in pair 9 of cell 33. Pairs 8 and 9 
clearly satisfy the conditions of the criteria and the remainder 
of the pairs duplicate the conditions in figures 33 and 34. 

The chromosomes of the cell represented by figures 24, 31, 
and 37 are foreshortened in nearly every case and were measured 


198 


CHARLES L. PARMENTER 


only in order to learn whether they constitute a series which 
would indicate that they belong to one cell. Only one set of 
measurements was made. Consequently, the figures are not so 
accurate as those of the other cells. Pairs 1 and 2 are readily 
recognized because they are well separated from each other and 
adjacent pairs. Pair 8 which stood out clearly in figures 33, 34, 
and 36, occupies a similar position here, but its homologues 
according to these less correct measurements differ about 4 mm. 
in length. The relative positions of the pairs practically dupli¬ 
cate those of the other cells. I have not attempted to make 
corrections for foreshortenings, but as nearly as I can judge, the 
chromosomes of pair 1, if corrected for foreshortening, would 
differ in length a little more, homologues of pair 2 would differ 
less in length, and the homologues of pair 8 are foreshortened 
about equally. Approximately the same condition exists in the 
other pairs, so that the matching as indicated would not be 
disturbed sufficiently to alter the grouping of the pairs. In 
this cell chromosomes of pair 12 differ by approximately 10 mm., 
which recalls a second time the condition in pair 9 of figure 33. 

Further consideration of the form of the chromosomes in all 
these figures furnishes additional strong evidence that the chro¬ 
matin is definitely organized. As indicated in anaphases, the 
general form of the chromosomes in the metaphase of these 
somatic mitoses is determined by the point of spindle fiber 
attachment. The complexes represented in figures 27 to 32 are 
early metaphases, and the final form which the chromosomes will 
take is quite apparent, although in some cases (e.g., pair 8, 
fig. 27) it is not entirely clear. 

In all of these figures, as previously mentioned, the form of 
the homologues of each pair is practically the same, even in 
cases where the final form has not been reached. Only three 
pairs (5, fig. 29; 7, fig. 30; 5, fig. 31) are exceptions, and this may 
be expected as indicated by a like condition in the heteromorphic 
pairs of certain Orthoptera (Carothers, T7). A comparison of 
several complexes from the same individual would probably 
show this condition constant for the individual as in the Or¬ 
thoptera. A further comparison of each pair in any figure with 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 199 


the corresponding pairs of all the other figures also shows a 
striking correspondence of form in these chromosomes. The 
homologues of the unusual cells represented by figures 31 and 32 
may not be correctly determined, as previously mentioned. 

Such agreement and constancy of form between homologues 
and between corresponding pairs of different individuals cannot 
well be considered as due to chance and indicates a definite 
organization of the chromatin. 

3. Summary. The strongest evidence for the existence of pairs 
is the fact that the chromosomes indicated as pairs 1, 2, and 8 
in figures 33 and 34 completely satisfy the criteria. Although 
these pairs fail to do so in figures 35 and 36, they are recognizable. 
Among those chromosomes composing the two large groups in 
which the chromosomes differ so little in length (pairs 3 to 7 
and 9 to 14) the evidence presented by the lengths of the chro¬ 
mosomes does not conclusively demonstrate nor deny the exist¬ 
ence of paii;s because of the various factors inherent in the 
nature of the material. However, as represented in the graphs 
and figures, the lengths and forms of these chromosomes strongly 
indicate such a duplexity. The cases in which the chromosomes 
of a pair differ somewhat in length do not constitute contrary 
evidence since homologues are not always of equal length as 
explained on page 217. Furthermore, the constancy of chromo¬ 
some number, the presence in all the cells of certain groups com¬ 
posed of the same number of chromosomes with approximately 
the same relative lengths is further strong evidence of a con¬ 
stancy of chromatin organization and that the lengths of the 
chromosomes are not due merely to chance. 

It seems to me to be a very difficult task to demonstrate 
conclusively by means of measurements the existence of pairs 
in these and similar somatic complexes. To furnish anything 
more than strong supporting evidence is almost impossible 
because of the various difficulties inherent in the nature of the 
material and because of the fact that homologues, as shown in 
exceptional cases, are not always of equal length, a fact which 
has been actually observed in the germ cells (tetrads) of certain 
Orthoptera by several authors. The same conditions make it 


200 


CHARLES L. PARMENTER 


just as difficult to demonstrate the absence of pairs. It seems 
to me that the evidence in the Dipteran somatic complexes, 
where the members of a pair lie parallel and adjacent to one 
another, together with the already large and well-supported 
evidence of pairs in the various generations of the germ cells 
throw the balance greatly in favor of the presence of homologues. 

DISCUSSION 

A. Introductory statement 

The foregoing observations upon the constancy of chromosome 
number and the existence of pairs in the somatic chromosome 
complexes have their chief importance in their relation to the 
Roux-Weismann hypothesis that the chromatin is the idioplasm, 
which is differentially organized and linearly arranged, and 
that this organization is perpetuated. This hypothesis received 
important support from the theory of the individuality of the 
chromosomes as set forth by Van Beneden (’83) and strongly 
maintained by Rabl (’85), Boveri (’88, ’02), and numerous other 
more recent investigators. The morphological evidence ac¬ 
cepted as supporting this proposition is an essential constancy 
of number, size, form, and behavior. 

Since McClung (’17) has so recently thoroughly considered 
the theory of individuality, this discussion is confined to the 
particular phases of the supporting evidence which are directly 
related to the observations made upon this material. These 
phases are essential constancy of number, of size, and of form. 

B. Constancy of chromosome number 

Della Valle has strongly attacked this theory on the basis of 
inconstancy of chromosome number. He arrives at the con¬ 
clusion (’09, p. 120 ff.) that the number of the chromosomes is 
the quotient of the quantity of the chromatin divided by the 
average size of the chromosomes; that their size is variable 
according to the nature of the elements and the conditions in 
which they are found, and (’ll, p. 188) that the size and number 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 201 

of the chromatic elements are directly comparable to the size 
and number of the fluid crystals which are formed in a solution 
under different conditions. 

These contentions he supports with the claim (’09) of a 
variation of nineteen to twenty-seven chromosomes in forty 
mitoses of the peritoneum, and (’ll) by a very large variation 
in the blood cells of Salamandra maculosa. These observations 
he supplements with a long list of citations of chromosome num¬ 
bers which he interprets as supporting his contention. 

But, following an apparently frank and critical discussion of 
the accuracy of his observations in the peritoneal cells, he says 
(’09, p. 116) that he is only sure of his enumeration in twenty- 
five of the forty cells discussed. These twenty-five complexes 
he describes as being very clear. The range of variation in these 
cells is as follows: 

Number of chromosomes.... 19 21 22 23 24 25 26 27 

Number of'mitoses. 1 1 1 6 16 12 2 1 total 40 

Number of mitoses. 1 1 3 10 8 2 total 25 

An examination of his descriptions and figures may indicate to 
some extent the reliability of these counts. 

His count of 22 chromosomes was made upon a polar view of 
a very late anaphase (fig. 2) in which he states the smaller chro¬ 
mosomes in the center of the complex were beginning to go to 
pieces and becoming indistinct, and his only doubt is whether 
the chromosome numbered 18 is one or two chromosomes. But, 
judging from similar stages in my material, it seems to me that 
where the chromosomes are beginning to go to pieces in as 
crowded a condition as this must be, such a complex is not a 
safe object for an exact chromosome count. 

Instances of this kind make it seem possible that conditions 
which he considers clear for an exact count might be much less 
conclusively clear to others, and that his drawings do not represent 
the actual conditions in his complexes. 

Since his citations of chromosome number variations found in 
the literature have been discussed by Montgomery (TO), Wilson 
(TO), and by McClung (T7, p. 548 ff.), it is not necessary to 




202 


CHARLES L. PARMENTER. 


review them extensively. But Della Valle’s attitude and his 
conception of what constitutes clearness and certainty may be 
better understood in the light of some of these citations of 
chromosome variations, especially in the Amphibia, which he 
presents as valid evidence of variation in chromosome number. 
He quotes (’09, p. 35) Flemming (’81, [’82] p. 51) and Rabl 
(’84, [’85] p. 248 to 250) as reporting variations of from seventeen 
to twenty-four in the gill epithelium of Salamandra maculosa. 
Flemming explicitly states (pp. 51 and 52) that in the three 
cells which admit an exact count there are twenty-four chromo¬ 
somes, that in about twenty other cells he counted from seven¬ 
teen to twenty-four, but was not certain of the number, and 
assumed that there were twenty-four. Rabl says (’85, p. 248) 
that up to that time only eleven unquestionable counts had been 
made and each of them showed twenty-four chromosomes. In 
no exact counts in any cell had a different number been found. 
Della Valle seems to think that Torok’s (’88) figures of erythro¬ 
cytes of Salamandra maculosa show a variation. This work 
was not concerned with chromosome number and the figures 
were not intended to show the number of chromosomes in the 
cell. His citations of the work of Carnoy and Lebrun (’00) on 
Rana temporaria, and of Lebrun (’02) on Diemyctylus and Bom- 
binator may be criticised because the authors were primarily 
concerned with other considerations and only gave approximate 
number determinations. Winiwarter (’00, p. 699), as cited by 
Della Valle, reports a variation of chromosome number in the 
rabbit; but he states that he is uncertain of his counts. The 
variations reported by Barratt (’07, p. 376), in proliferating 
epithelium of the rabbit are in pathological tissue, and, more¬ 
over, his counts are uncertain. Montgomery (’10) has shown 
that many other such citations are misinterpreted. 

The above cases represent Della Valle’s inexact and uncritical 
attitude in relation to data that seem to serve his purpose, and 
this creates the suspicion that his attitude interfered with the 
accuracy of his observations when he counted the chromosomes 
in his own material. This suspicion approaches a probability 
in view of the fact that numerous competent investigators who 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 203 


have made extended and careful studies of the germinal and 
somatic mitoses of the same species mention no variation in 
chromosome number. Furthermore, Meves (’ll), who attacks 
the theory of individuality, fails to substantiate Della Valle’s 
observations, and it is not at all likely that he would have failed 
to mention any variation observed. He appears to believe (p. 
296) that the number is constant. However, Heidenhain (’07, 
p. 176, figs. 80 and 81) shows a polar view T of a late prophase 
and a lateral view of a metaphase with twenty-six and twenty- 
two chromosomes, respectively, and states that such irregu¬ 
larities occasionally occur. An occasional variation is not sur¬ 
prising, but variations as numerous as Della Valle claims to be 
present are unusual. Flemming (’90, p. 78) states that he 
observes in the lungs of Salamandra maculosa numerous atypical 
rqitoses with very short chromosomes. He gives no further 
discussion and no figures to indicate what kind of cells they are 
nor whether they are normal. In the ten cells of the lung of 
Ambystoma tigrinum (table, p. 177) there were no variations in 
chromosome number, and with the exception shown in figure 26 
I observed no abnormalities. Della Valle’s (’ll) figures of blood 
cells in Salamandra maculosa, which he claims show an extreme 
variation in chromosome number, appear very much like cells 
undergoing disintegration. 

To the above evidence of the questionableness of Della Valle’s 
results may be added the results of the sixty-six counts in Amby¬ 
stoma tigrinum showing no variation in number. The important 
fact that these counts were made with extreme care (p. 177) in 
the somatic cells of the same and other tissues of a closely related 
species, and made in uncut membranes (which Della Valle 
emphasizes as important for accurate counts), further strengthens 
the already strong probability that his number determinations 
are incorrect. 

There are certain characteristics in his figures that also indicate 
that his drawings are none too accurate. He notices that the 
chromosomes are twisted, but he does not show what constitutes 
the twist. That the peritoneal chromosomes of Salamandra 
maculosa are each composed of two separate chromatids twisted 


204 


CHARLES L. PARMENTER 


about one another is plainly evident in Meves’ (’ll) figures 11 
to 15 which show each chromosome to be of variable width. 
These figures are exactly comparable to my figures 1 to 8, and 9 
to 23 which demonstrate that this variation in width is due to 
the twisting of the chromatids. Della Valle represents each 
chromosome to be of uniform width excepting an occasional 
split in the end of some chromosomes. If he does not see chro¬ 
matids in any of the chromosomes which he has drawn, either 
his observations, his technique, or both are faulty. Further¬ 
more, the above evidence together with his attitude make it 
uncertain whether his preparations were as clear or the chromo¬ 
somes as distinctly separated from one another as his drawings 
indicate. 

Finally, Della Valle’s above demonstrated attitude, the ab¬ 
sence of confirmatory evidence for his contentions, his question¬ 
able ability as an observer as indicated by his drawings, and the 
results of critical counts in Ambystoma tigrinum, all support 
the view that his observations and conclusions are incorrect. 

But upon the assumption that they may be partially correct, 
there are some possible explanations for the presence of variation 
in the peritoneum of Salamandra maculosa. 1) One or more 
chromosomes of a complex easily could have been disturbed, as 
is evident from my figures 22 and 24 to 26. This could account 
for number deficiencies and perhaps also for excesses. 2) 
Champi (T3, p. 181) claims that chromosome number can vary 
by fragmentation under the influence of certain external stimuli. 
Della Valle (’09, p. 86) says the number of mitoses can be in¬ 
creased by keeping the larvae covered with a blue glass. If 
Della Valle did this, and if such a stimulus could produce frag¬ 
mentation, a bare possibility is offered for a disturbance of 
chromosome number. 3) There is also a slight possibility that 
the larvae had been kept in captivity and might in consequence 
have been sufficiently pathological to produce abnormal mitoses. 
4) In an investigation on certain Orthoptera now in progress in 
this laboratory, Mr. Carroll observes that in three individuals 
some of the few dividing spermatogonial cells contain, in ad¬ 
dition to the normal number of twenty-three, one, and some two 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 205 


extra chromosomes (dyads). This is a small variation in chro¬ 
mosome number in the individual of from twenty-three to twenty- 
five. In each of four individuals, including the above three, he 
finds that in from one to three of the primary spermatocytes 
observed in division, one of the eleven tetrads normally present 
is replaced by two separate dyads. Both of these dyads in the 
division of the cell may pass undivided to either daughter cell 
with or without the accessory chromosome. One of the secondary 
spermatocytes resulting from the division of the cell in which 
these two dyads accompany the accessory chromosomes receives 
twelve dyads plus the accessory and the other receives ten 
dyads. The other spermatocyte in which two dyads do not 
accompany the accessory chromosome gives rise to one secondary 
spermatocyte with ten dyads plus the accessory, and another 
with twelve dyads. This would make possible four classes of 
spermatozoa containing ten to thirteen chromosomes. 

If, similarly, in Salamandra maculosa one of the twelve 
tetrads normally present in the spermatocytes and in the oocytes 
should be replaced by two dyads, there would be produced 
gametes with eleven, twelve, and thirteen chromosomes. These 
gametes would produce zygotes (individuals) having twenty-two 
and twenty-six chromosomes, respectively, a variation com¬ 
parable to that claimed by Della Valle. Further, if extra chro¬ 
mosomes can thus appear in the germ cells of an occasional 
individual, the same might also occur in the somatic cells. But 
this variation should be expected in but few of the total indi¬ 
viduals, making the proportion of cells containing the normal 
number greatly predominating. In Della Valle’s counts about 
one-half contain the normal number which is far too small a 
portion unless such variation is much more common than is at 
present known. 

Although the above is a clear case of a small variation in chro¬ 
mosome number in the individual , it must be clearly understood 
that these cases are exceptional and do not represent the normal 
condition. The chromosome number may vary in the species, 
but it is usually constant for the individual, as has been 
especially pointed out by Wilson (’09, TO), Carothers (’17), 


206 


CHARLES L. PARMENTER 


McClung (’05, ’07, and ’17), and others. But it is very im¬ 
portant to note that these irregular chromosomes arise and 
perpetuate themselves in a manner entirely consistent with a 
definitely organized chromatin and furnishes no support what¬ 
ever for Della Valle’s contention that the chromosomes are 
comparable to crystallizations of a salt solution and that their 
number in any cell is dependent upon the law of chance. 

C. Variations in other Urodeles 

Snook and Long (’14) find in the spermatogonial cells of 
Aneides lugubris nine containing clearly the usual number of 
twenty-eight chromosomes, and one cell with twenty-three. 
There are no other authentic reports of variable chromosome 
number in individuals of the Urodeles. The counts of Kolliker 
(’89), Fick (’93), and Jenkinson (’04) in the cleavage stages of 
Axolotl (Ambystoma tigrinum) were made for other purposes, 
and were not presented as accurate number determinations. 
Likewise, the counts of about eighteen to twenty-four, Champi 
(’13, p. 124) in several other Salamanders are only approximate. 

D. Variations in other forms 

Since a somewhat extensive tabulation of comparative germi¬ 
nal and somatic counts has been made and discussed by Hoy 
(’16), Harvey (’16), and briefly reviewed by Hance (’17 b), a 
repetition of this discussion is of little value. However, in the 
review of reported cases of variation a few general considerations 
have impressed me as worthy of mention. These may seem 
commonplace, but are evidently not altogether realized by some 
who are none too critical in their discussion of the significance 
of these enumerations. 1) As Montgomery (’01) long ago sug¬ 
gested, it is important to distinguish between variation in the 
chromosomes in the germinal line and those of differentiating 
somatic cells. In the germ cells I believe it can be stated safely 
that there are no certainly demonstrated variations in number 
which do not conform to a definite organization of chromatin. 
From time to time cases of apparent variation have appeared 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 207 


and again disappeared when thoroughly understood (e.g., Meta- 
podius Wilson, ’10, and the sex group of Ascaris lumbricoides 
Edwards, ’10, and multiple chromosomes of certain Orthoptera 
Woolsey, ’15, Robertson, ’16, and McClung, ’05, and ’17). 2) 

In considering the significance of variations, it should be remem¬ 
bered that there are normal and abnormal conditions (p. 202). 
3) Metz (’16), Hance (’17 a, b), and Whiting (’17) have called 
attention to the necessity of proper technique. This is not 
always an easy matter to judge, especially in absence of material 
for comparison. 4) In determining the presence or absence of 
variation in any material, a very rigid line should be drawn 
between accurate enumerations and those involving varying 
degrees of interpretation, e.g., Winiwarter’s (’00) cited varia¬ 
tions in the amnion and omentum of rabbit embryos were uncer¬ 
tain and interpreted. 5) Finally, observers should maintain an 
exacting standard in distinguishing between that which is con¬ 
sidered 'certain’ and that which is interpreted. This is 
especially true in counting small chromosomes. 

E. Fragmentation 

Hance (’17 b, ’18 a) found the spermatogonial number in the 
pig, and the diploid pollen-mother cells in Oenothera scintillans 
to be constantly forty and fifteen, respectively. But the somatic 
chromosomes vary from forty to fifty-seven in the pig and from 
fifteen to twenty-one in Oenothera scintillans. He presented 
metrical evidence that this variation is due to a fragmentation, 
probably of the longer chromosomes. He maintains that these 
fragments divide normally with the other chromosomes, and 
that therefore this fragmentation does not oppose the theory of 
the individuality of the chromosomes. 

However, the probability that this variation in Oenothera is 
much less, and that most if not all of these fragmentations are 
invisibly connected with the main part of the chromosome is 
strongly supported by the conditions in Ambystoma material 
and by Hance’s (’18 b) later observations upon additional 
Oenothera material from the same source. Both he (’17 b, p. 90) 


JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 


208 


CHARLES L. PARMENTER 


and Hoy (’16, p. 356) review other cases of fragmentation in 
Ascaris megalocephala (Boveri, ’99, ’04), Angiostomum (Schleip, 
’ll) and Fragmatobia (Seiler, ’13). 

In Ambystoma tigrinum (p. 179) and in Salamandra maculosa 
(Della Valle, ’09, fig. 11), and as stated above, in Oenothera, the 
fragmented portions are directly in line with the main portion 
of the chromosome. This may be due to the absence of chro¬ 
matin on the linin or failure of the chromatin to stain at that 
point. The fact that in several cases (e.g., /, figs. 5, 6 and 7) 
the space between the fragment and the main portion of the 
chromosome was uniformly faintly stained lends support to the 
suggestion. Other cases exhibited connections consisting of 
various amounts of strongly stained chromatin (e.g., chr.f., figs. 
5, 14, 15, and 21). All mitoses in the gill plates (the most in 
prophases, fig. 5) showed the largest number of instances of this 
condition; the peritoneum contained scarcely any. This might 
be explained as an effect of inferior fixation (p. 173) were it not 
for the fact that a considerable amount of apparent fragmenta¬ 
tion is present, even in the metaphases of the tail epithelium 
which are fixed under the most favorable conditions. The 
reason for this is not clear. 

F. The existence of pairs 

The question whether the chromosomes exist in a duplicate 
series is significant in two respects: 1) in its relation to the 
mechanism of heredity as suggested by Janssen’s chiasmatype 
theory and by the brilliant work of Morgan and his co-workers; 
2) as a further index of the constancy of the organization of 
the chromatin. This constancy is vitally related to the theory 
of the individuality of chromosomes. 

It will be convenient to consider separately the evidence of 
the existence of pairs in the germ cells and in the somatic cells. 

a. Pairs in germ cells. Van Beneden’s (’83) hypothesis, that 
one-half of the chromosomes of an individual are of maternal 
origin and that the other half are of paternal origin, has been 
verified in many cases. That this double set of chromosomes 


CHROMOSOME* NUMBER AND PAIRS IN AMBYSTOMA 209 


exists in pairs in the germinal line is evidenced by their behavior 
during the maturation period. 

Montgomery (’01) presented evidence for the recognition of 
pairs in the spermatogonia, basing his argument upon the signifi¬ 
cance of the chromosome number in Euschistus. Sutton (’02) 
showed by means of a comparison of many camera-lucida draw¬ 
ings of spermatogonial complexes of Brachystola magna that 
these chromosomes form a duplicate series of lengths, and by 
means of measurements with a pair of dividers that the chro¬ 
mosomes of the early primary spermatocyte prophases are graded 
into the same series of relative sizes. Meves (’ll) interpreted 
his measurements upon spermatogonia of Salamandra maculosa 
as failing to demonstrate pairs. Meek (’12) has made linear 
measurements upon the spermatogonia and secondary spermat¬ 
ocytes of a number of animals, interpreting his results as con¬ 
firming the claim of the existence of pairs. Robertson (’15, ’16) 
also supports this view with metrical data in certain Orthoptera, 
and Hance (’17 b; ’18 a) confirmatively interprets his measure¬ 
ments in the germinal and somatic cells of Oenothera and the 
pig. In unmeasured spermatogonial chromosomes of the Dip- 
tera, Stevens (’08, ’10, ’ll), Metz (’14, ’16), and Whiting (’17) 
show very convincing evidence of pairs for the homologues are 
associated side by side. Wilson’s (’06, p. 11) figures of Anasa 
and Hoy’s (’16, p. 336; ’18) figures of Anasa, Epilachna, and 
Diabrotica also support this conclusion. The majority of other 
authors as a result of their general observations have expressed 
the belief that the chromosomes exist as a duplicate series. 

Furthermore, the existence of pairs in the spermatogonia is 
practically proved by parasynapsis where the chromosomes of 
the last spermatogonial division unite side by side and remain 
so until separated by the reduction division. This statement is 
made possible by Wenrich (’16) who, besides confirming the 
already numerous and all but conclusive evidences of parasynapsis 
by Janssen (’05, ’09), A. and K. E. Schreiner (’06, a and b; 
’08, a and b), Wilson (’12), and many others, carries the demon¬ 
stration a step further by actually tracing a well-marked chro¬ 
mosome pair A (p. 76) continuously through every stage of 


210 


CHARLES L. PARMENTER 


spermatogenesis from the early spermatogonia to the spermatids. 
He thus demonstrates that the conjugating elements are chro¬ 
mosomes and are morphologically identical with the spermat- 
ogonial ’chromosomes. That one of the homologues of each 
conjugated pair is maternal and the other paternal is very 
probable, as has been shown by the observations of Van Beneden 
(’83) and numerous later authors, especially Mulsow ( , 12). 

It remains to be seen whether the pairs of maternal and 
paternal homologues present in the germ cells during the matura¬ 
tion period maintain their identity in the germinal line between 
the time of fertilization and the first observations upon the sper¬ 
matogonia. This has been accomplished in part. Mulsow (T2) 
has followed the actual chromosomes of the living spermatozoon 
of a parasitic trematode, Ancyracanthus, into the egg, and has 
found the expected number of chromosomes in the two pro¬ 
nuclei and cleavage stages. He also observes that the chromo¬ 
somes of the cleavage nuclei show in many cases a tendency to 
lie parallel to one another, and suggests that this is an approxi¬ 
mation of maternal and paternal chromosomes. Boveri (’87, 
J 92) traced the chromosomes of the primordial germ cell of Ascaris 
univalens through the cleavages from the two-celled stage, 
and Moenkhaus (’04), Morris (T4), Richards (T7) have traced 
the persistence of individual chromosomes through several 
cleavages of hybrid eggs of Fundulus. If this persistence of the 
chromosomes is permanently maintained, the observations of the 
above authors make it probable that the maternal and paternal 
chromosomes form a duplicate series throughout the germinal 
line. 

b. Pairs in somatic cells. Since the existence of chromosome 
pairs can be considered to be all but proved throughout the 
germinal line, it remains to be seen whether or not the chromo¬ 
somes of the somatic cells, which are really descendants of those 
of the germinal line, still retain this duplicate series and thus give 
evidence of maintaining their individuality. 

The earliest observations bearing upon this question were 
made on Salamandra maculosa by Flemming (’82) and Rabl 
C85), who observed that the chromosome segments were not of 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 


211 


equal lengths in the early spireme and metaphase stages. The 
general observations of many later authors, especially those 
studying Dipteran somatic cells (Metz, T4, T6 a, b; Iiance, T7; 
Holt, T7; Whiting, T7) indicate these chromosomes to be paired. 
Observations of pairs in a large number of animals and plants 
have been extensively reviewed by Metz (T6, p. 245). 

But no cases are recorded in which an attempt was made to 
determine accurately by measurement what relation the lengths 
of somatic chromosomes bore to one another until the work of 
Meves (’ll). As a result of the discussion centering around the 
observations of Montgomery (’01) and Sutton (’02), he was led 
to attempt measurements in an effort to obtain more definite 
data, as suggested by Della Valle (’09, p. 109). He measured 
both spermatogonial and somatic chromosomes of various tissues 
in Salamandra maculosa. Della Valle (’12) made further meas¬ 
urements upon the same form, and agrees with Meves that their 
results do not confirm the observations of Montgomery and 
Sutton. Hance (’17 and T8 a) interprets his measurements 
upon the somatic chromosomes of Oenothera scintillans and the 
pig as confirmatory. • 

1. Meves’ results. Since Meves has (p. 282) failed to confirm 
the results of other authors, it is desirable to reconsider his data 
in comparison with linear measurements upon cells of the same 
nature in the same kind of preparations and of the same tissues 
of another salamander, Ambystoma tigrinum, in an effort to form 
a judgment of the validity of’his conclusions. For this purpose 
it is necessary to recall what criteria are required (p. 191) 
definitely to affirm or to deny the existence of pairs and under 
what conditions these criteria were satisfied. 

The following conditions under which Meves’ measurements 
were made allow the introduction of such a varying amount of 
error that the conclusions drawn from his results are of question¬ 
able value. 

Instrumental and personal errors. Meves made his measure¬ 
ments evidently upon a single drawing, probably somewhat care¬ 
fully executed, which means, according to a series of tests in my 
own attempts to be accurate, that he has a minimum instru- 


212 


CHARLES L. PARMENTER 


mental and personal error of about 0.6 mm. at his magnification. 
This, of course, is negligible in comparison with the large errors 
arising from foreshortening in numerous chromosomes. 

Concerning the favorableness of the spermatogonial chromo¬ 
somes for measurement, Meves says (p. 274) that by no means 
do all of the chromosomes lie in the equatorial plane; without 
exception the bend lies in the plane while the ends lie outside; 
in the drawings such chromosomes seem shortened and therefore 
the measurements upon these chromosomes would give only an 
approximate value. Judging from these statements and from 
the magnitude of error due to the slight foreshortenings in my 
material, his measurements very likely contain errors which 
amount to as much as 4 or 5 mm. 

For measurements of somatic chromosomes he chose (p. 280) 
polar views of the transformation stages between the prophase 
and metaphase stages in the epithelium of the gill plates (figs. 16 
to 18) and extraordinarily well-flattened polar views of pro¬ 
phases (figs. 11 to 13) and metaphases (figs. 14 to 15) in the 
peritoneum. In the three prophases of the peritoneum the chro¬ 
mosomes lay nearly or entirely parallel with the upper surface 
of the cell. According to this description, it is evident that these 
three prophases are the most favorable cells, and even in these 
the chromosomes are not entirely free from foreshortening. The 
chromosomes of the other cells probably were more foreshortened. 
Therefore, judging from results in Ambystoma, his measurements 
contain errors due to foreshortening which probably vary from 2 
to 5 mm. 

The amount of the errors which are due to the twisting of the 
chromatids of these chromosomes is uncertain. Such twisting is 
evidently present, as indicated by the irregular contour of his 
chromosome drawings which are similar to those of my own. 
The errors due to this twisting may largely neutralize each other 
as explained above (p. 189). He mentions also the possibility of 
different rates of contraction of the chromosomes, especially in 
the earlier stages. Having before us the conditions under which 
Meves made his measurements, we are in a position to judge 
their value more or less correctly. 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 213 


As Meves states (p. 276), the differences in length between the 
spermatogonial chromosomes are too small, and the possible 
errors too great to affirm that these chromosomes are present in 
pairs of equal length. On the other hand, it should be added 
that these conditions offer no evidence against this claim. 
Furthermore, he shows a practically constant difference in 
length of 6 half-millimeters between chromosomes 8 and 9 in 
his table of measurements. An examination of these figures 
shows unmistakable evidence that there is foreshortening in chro¬ 
mosome 9 in three of these cells, and a probability that chromo¬ 
some 8 is much less, if any, foreshortened than chromosome 9. 
There results, therefore, in all of these cells a relatively uniform 
distribution of the chromosomes into two constant groups, a 
point which supports the claim that the relative lengths of these 
chromosomes remain constant. 

Concerning the somatic measurements, Meves concludes (p. 
282) “Die Fragen, ob bei gewohnlichen Gewebszellen des Sala- 
mandra Chromosomenpaaren unterschieden werden konnen, ist 
bereits von C. Rabl (’06, S. 72) verneint worden, ich muss mich 
ihm auf Grund der mitgeteilten Zahlen anschliessen” and “Die 
erhebliche Langendifferenz zwischen chromosomen VIII und 
IX, welche wir bei den Spermatogonien festgestellt haben, 
besteht bei den gezeichneten somatische Zellen nur in der Halfte 
der Falle.” This means, of course, that he believes that the 
chromosomes are not distributed into two constant groups in 
each cell and that therefore the evidence of the constant organi¬ 
zation of the chromatin is lacking in this respect. 

As already stated, the possibilities of error are so great that 
nothing is conclusively affirmed or denied by his measurements. 
However, an examination of the drawings of the chromosomes in 
his figures indicates certain probabilities. 

1. In three of the four cells (his figs. 15 to 17) in which the 
marked difference between chromosome 8 and 9 is not present, 
chromosome 9 is conspicuously foreshortened, and is in reality 
longer than his measurements indicate. Furthermore, chromo¬ 
some 8 in these cells may or may not be foreshortened, at any 
rate it is probably much less foreshortened than chromosome 9. 


214 


CHARLES L. PARMENTER 


A conservative estimate of foreshortening upon chromosome 9 in 
these cells, based upon computation of foreshortening in my 
material, amounts to a minimum of 2 mm., which restores in 
these cells the difference in length between chromosomes 8 and 9 
found in the spermatogonial and the other somatic cells. These 
same chromosomes in the remaining cell (fig. 13) which lack this 
difference do not appear foreshortened, but owing to the various 
sources of error pointed out in the preceding pages, it is entirely 
possible that such a difference may also be present in this cell. 
These considerations make it probable that the difference be¬ 
tween chromosomes 8 and 9 is present in eleven and possibly in 
all of the twelve somatic cells which Meves measured. It is 
beyond expectation that this difference should be exactly the 
same in every cell whether of the same or of different stages of 
mitosis. 

2. Meves also observes a difference of 3 to 4 mm. between 
other chromosomes which theoretically should be homologues. 
In figure 11 between chromosomes 17 and 18 and between 
chromosomes 23 and 24; in figure 12, between chromosomes 11 
and 12; in figure 13, between chromosomes 7 and 8, 15 and 16, 
17 and 18, 23 and 24. 

Attempts to apply corrections for the foreshortening evident 
in these cells, estimated upon the basis of his drawings as com¬ 
pared with similar cells in Ambystoma (figs. 5, 13, and 14), 
leave the situation about as it was. This is not surprising, con¬ 
sidering the difficulty of judging the amount of foreshortening 
that is conspicuously present in some chromosomes and the 
uncertainty of its presence, or absence, in others. Meves’ state¬ 
ment quoted above concerning the type of cells used for measure¬ 
ments makes it quite possible that a great many of these chro¬ 
mosomes are foreshortened in amounts varying from 2 to 5 mm. 
This opinion is strengthened by his generalized and unprecise 
statement concerning foreshortening and by comparisons with 
similar cells in Ambystoma. 

The conspicuous foreshortening in some chromosomes, and 
the probability of it in others, seriously weakens the validity of 
his measurements. This is especially true of figures 11, 12, 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 215 


and 13 showing late prophases in which there is usually much 
foreshortening and considerable inequality of homologues, as 
stated above. In five other cells (figs. 14 to 18) Meves’ figures 
show but one case of a difference of 3 mm. and only five cases 
of a difference of as much as 2.5 mm. between homologues, as 
they are indicated by his measurements. But the various 
sources of error already mentioned make it uncertain as to what 
the actual lengths are. 

To summarize the examination of the results of Meves’ 
measurements, it may be said, 1) that the chromosomes of the 
spermatogonial cells fall into two groups, one containing chro¬ 
mosomes 1 to 8, the other chromosomes 9 to 24. 2) In the 

somatic cells, when corrections are made for evident fore¬ 
shortenings, it is probable that in eleven of the twelve cells, and 
possibly also in the twelfth, the same grouping is present. This 
indicates a constancy of organization of the chromatin. 3) It 
is impossible, either to demonstrate conclusively or to deny that 
these chromosomes are paired because, a) of the various sources 
of error present and, b) the small differences in length in the 
majority of cases between adjacent chromosomes. 

2. Della Valle’s measurements. Della Valle (’12) measured 
the lengths of chromosomes in the peritoneal cells of Salamandra 
maculosa shown in his (’09) figures 1 to 3, 8 to 9, and 12. The 
length of each chromosome was obtained by averaging two 
measurements made with a curvimeter upon a single camera- 
lucida drawing. He also attempts to determine the degree of 
concordance between the measured lengths of each of these 
chromosomes and the dimensions which would exist if the 
lengths of these chromosomes were determined by the laws of 
fluctuating variation. These latter figures he obtains by calcu¬ 
lation from a table of figures compiled by Sheppard and pub¬ 
lished by Galton (’07). He interprets his data as demonstrating, 
1) that the chromosomes of Salamandra maculosa do not exist in 
pairs; 2) that there is no constant grouping of chromosomes, 
such as is evident in the measurements of Meves and myself, 
and, 3) that the chromosomes are a series of variants subject 
to the laws of fluctuating variation as shown by the comparison, 


216 


CHARLES L. PARMENTER 


given in his tables and curves, between the measured lengths of 
the chromosomes and the computed lengths that would be 
expected if the chromosomes were such a series of variants. 

I believe Della Valle’s conclusions are incorrect for the fol- 
owing reasons: 1) He fails to demonstrate the presence of 
chromosome pairs because, a) as discussed on page 201, his chro¬ 
mosome enumerations are probably incorrect and therefore his 
measurements do not represent the actual conditions; b) his 
measurements probably contain numerous errors of varying 
magnitude due to foreshortening (as well as to errors arising 
from measurements upon single drawings) even though he chose 
for measurements strongly flattened cells (p. 126); c) the dif¬ 
ferences in the lengths of these chromosomes are so small and 
the errors so great that it is impossible either to demonstrate or 
to deny a presence of pairs. 2) Failure to find a constant 
grouping among the chromosomes would result from the causes 
given in (a) and (b). 3) His interpretation that the chromosome 

lengths are controlled by the law of fluctuating variations is 
untenable because, even if his measurements were reliable and 
whether pairs do or do not exist, the differences in length between 
the chromosomes of Salamandra maculosa are so small that the 
degree of correspondence between their measured lengths and the 
calculated lengths of a series of variants, corresponding respec¬ 
tively to each of these chromosomes, would be fully as close as 
those which he presents in his tables and curves containing 
numerous and large differences. 

3. Results in Ambystoma tigrinum. The chromosomes of Am- 
bystoma tigrinum, fortunately, are more favorable subjects for 
measurements than those of Salamandra maculosa, because the 
relative differences in length between many pairs is so large 
that certain pairs and certain groups of pairs stand out con¬ 
spicuously. The evidence presented in figures 33 and 34 is free 
from all errors of measurement except those due to twisting of 
the chromatids and to minute foreshortenings at non-critical 
points in the series. These errors have been approximately 
eliminated (p. 188) and do not seriously disturb the critical evi¬ 
dence of the chromosome pairs which are much shorter or longer 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 217 


than adjacent pairs. The evidence in the other figures is but 
little inferior to that of figures 33 and 34. However, measure¬ 
ments of the chromosomes of so few cells are insufficient to 
furnish more than strongly supporting evidence of the existence 
of pairs, and of an approximate constancy of size relations 
between pairs in different individuals. 

It may appear that these measurements support equally well 
Della Valle’s claim that there are no chromosome pairs, but that 
the chromosomes form a series of variants. However, the con¬ 
sistent evidence of the presence of pairs among the shorter 
chromosomes, the possibility of unequal stretching of the longer 
homologues together with the known condition in Orthoptera 
that homologues of tetrads may be of unequal lengths lends 
greater support to the probability of the existence of homologues. 

The following points need further consideration. 

Large differences in length between homologues . . The difference 
in length of 12.4 mm. between the homologues of pair 9 in 
figure 33 and the similar difference of 9.5 mm. between the 
homologues of pair 12 in figure 37, the smaller difference in 
pairs 7 and 8, figure 35, pair 7, figure 36, and pair 8, figure 37, 
may possibly be explained as follows: 

1. Unequal homologues have been reported in Orthoptera by 
Baumgartner (’ll), Hartmann (’13), and more thoroughly studied 
by Carothers (’13), Robertson (’15), and Wenrich (’16). The 
latter’s observations are particularly significant. He found dif¬ 
ferent conditions of inequality in two of the small tetrads of 
Phrynotettix. He designated these two tetrads as ‘B’ and 
‘C’ and traced their history from the pachytene stages through 
the first maturation division. The homologues of tetrad ‘B’ 
were unequal in eleven of the thirteen individuals studied and 
were equal in the other two. Tetrad ‘C’ was found in three 
forms, designated as ‘Ci, C 2 , C 3 .’ ‘Ci is composed of very 
unequal elements, the larger of which possesses a relatively large 
terminal knob or granule which is not present on the other two. 
‘C 2 ’ is a pair with equal members, each of which appears to be 
homologous to the smaller member of ‘Ci.’ ‘C 3 ’ is a pair of 

unequal elements, neither member of which appears to be 


218 


CHARLES L. PARMENTER 


exactly homologous to the components of ‘CV and ‘C 2 .’ The 
smaller member resembles those of ‘ C 2 ’ and may be homologous 
with them. It is important to note that the three last-mentioned 
authors find that each particular condition is constant for the 
individual in which it is found. 

Although tetrads with unequal homologues among the longer 
chromosomes have not been observed in the Orthoptera, they 
might possibly exist in other animals. The above observations, 
especially those of Wenrich, offer a possible explanation for the 
inequalities between homologues observed in Ambystoma as well 
as in Salamandra maculosa. Furthermore, the condition found 
in tetrad ‘C 2 ’ may offer a parallel explanation for the different 
relative lengths shown in some cases between corresponding 
pairs in complexes of different individuals (e.g., pr. 4 and pr. 
9). Of course much further data from both the somatic and 
germinal chromosomes is necessary before the above can amount 
to anything more than a suggestion. 

2. Certain inequalities might be explained as due to the 
presence of a multiple chromosome similar to that which has 
been described by McClung (’05, T7), in Orthoptera, by Boveri 
(’09), Edwards (TO, Tl), and Frolowa (T2), for Ascaris megalo- 
cephala, Boveri (Tl) and Edwards (Tl) for Ascaris felis, Stevens 
(Tl) in Anopheles, and by King (T2) for Necturus. In these cases 
the sex chromosome has been interpreted as being attached to one 
of the euchromosomes, and thus there is present in the male an 
unequal pair of chromosomes which may parallel the condition 
in pair 9 of figure 33 and pair 12 of figure 37. In certain Or¬ 
thoptera McClung (T7) finds that the accessory may be attached 
to different chromosomes in different individuals, which lends 
support to the possibility that this is the condition in Ambystoma. 
The presence of an X and a Y chromosome would also produce 
unequal homologues. 

If either of these explanations be valid, such an unequal pair 
should appear in all the diploid complexes of approximately 
one-half of a somewhat large number of individuals, and similar 
conditions should be found in the maturation period. Unfor¬ 
tunately, the difficulty of obtaining a sufficient number of suitable 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 219 


cells prevents extensive investigation of this point in the somatic 
cells for the present and makes this explanation only suggestive. 

Constancy in the individual. It should be emphasized that the 
observations in the Orthoptera concerning the unequal tetrads 
(p. 217) and other heteromorphic tetrads (p. 192) strikingly 
demonstrate a constancy in the individual for the particular 
characteristic of each homologue concerned. In Ambystoma it 
has been impossible to obtain sufficient material to verify this 
point. 

Whether the members of these Orthopteran unequal and other 
heteromorphic tetrads maintain their organization from one 
generation of animals to the next is yet to be demonstrated by 
breeding experiments now in progress in this laboratory. The 
expectation is that they do, since Wenrich (T6) and Carothers 
(’17) find every possible combination which would arise from the 
segregation and recombination of the members of these various 
types of tetrads. 

The presence in the Orthoptera of unequal tetrads does not 
indicate a lack of individuality. On the contrary, the per¬ 
sistence of this condition throughout the individual, and perhaps 
from generation to generation, is strong evidence to the con¬ 
trary. Of course a change has taken place at some time (if it 
be correct to assume that the homologues were all alike at some 
earlier period), but this is to be expected if these chromosomes 
are to parallel genetic behavior. 

Bridges (’17, p. 445-6) presents parallel genetical data in 
connection with the chromosomes of Drosophila. He finds in 
certain cases that the genes for ‘bar’ eye and ‘forked’ bristles, 
whose loci are located near one end of the sex-chromosome, have 
been lost and that the region between these two loci has also 
been affected. He suggests that this deficiency may be due to 
a physical loss of this portion of the chromosome. He also 
reports (T9, p. 357) a case in which “a section of the X-chromo- 
some, including the loci for vermilion and sable, became 
detached from its normal location in the middle of the X-chro- 
mosome and became joined on to the ‘zero’ end (spindle fiber) 
of its mate.” In other instances the locus for sable alone, as 


220 


CHARLES L. PARMENTER 


far as known, has been lost from one homologue and joined to 
the end of its mate. Another case is the transposition of a piece 
of the II chromosome to the middle of the III chromosome. 
He has exhibited definite cytological evidence (unpublished) 
supporting a part of the above. This condition produces 
homologues of unequal length which parallels the observations 
in the germ cells of the Orthoptera and the apparent similar 
condition in certain somatic homologues of xVmbystoma. 

c. Constant relative size relations. In addition to verifying 
Montgomery’s (’01) and Sutton’s (’02) observations of paired 
homologous chromosomes of equal length in the germ cells, 
Meek (’12), Robertson (’16), and Hance (’17 b, ’18 a) confirm 
Sutton’s (’02) observation (based upon comparisons of camera- 
lucida drawings of many spermatogonial cells and upon measure¬ 
ments of early prophase tetrads) that the proportional difference 
in size between any two pairs in one nucleus is practically the 
same as that between the corresponding pairs in any other 
nucleus. In Ambystoma tigrinum, as is seen in figures 33 to 
37 and the table of percentages accompanying them, while 
the relative lengths are not exactly the same in every cell, there 
is in general a marked constancy of relative lengths. Were 
Meves’ and Della Valle’s measurements correct, the same would 
probably appear there. 

d. Summary of measurements. The. data here presented in 
connection with measurements upon the chromosomes of Amby¬ 
stoma tigrinum and Salamandra maculosa cannot well be inter¬ 
preted as a confirmation of Meves’ and Della Valle’s contention 
that pairing of the chromosomes and a constant organization of 
the chromosomes do not exist because: 1) Their data, on 
account of errors inherent in the material, are too unreliable to 
command confidence; 2) the differences in the lengths of the 
chromosomes of Salamandra maculosa are too small to permit 
one to deny or to affirm the existence of pairs, and, 3) it has 
been shown that because of obvious foreshortenings, individually 
mentioned above, which are unaccounted for in Meves’ measure¬ 
ments, there probably is a somewhat constant difference between 
chromosomes 8 and 9 in eleven out of the twelve cells which he 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 221 


measured. This confirms the contention of a constancy of 
chromatin organization so far as is possible in material having 
chromosomes differing so little in length as those of Salamandra 
maculosa. 4) The measurements of chromosomes in the so¬ 
matic cells of Ambystoma tigrinum show duplication of sizes, 
especially pairs 1, 2, and 8 in figures 33 and 34, pairs 8, 9, and 10 
in figure 36 and indications of the same in the other chromo¬ 
somes of all the cells which differ too little in length to constitute 
reliable evidence. Explanations are offered for cases in which 
the homologues differ in length. 5) The chromosome lengths 
show approximately constant relative sizes in all of the cells 
measured. 

Based upon the above considerations and upon the unequal 
and other heteromorphic tetrads in Orthoptera, my expectations 
are that the pairs and their relative lengths in the somatic cells 
of Ambystoma are constant for the individual, and although 
not exactly jthe same, they are approximately the same in dif¬ 
ferent individuals. However, as stated above (p. 199), the meas¬ 
urements cannot be considered to demonstrate conclusively the 
presence of a duplicate series of chromosomes. 

SUMMARY OF CONCLUSIONS 

1. No variation is found in the somatic chromosome number 
of twenty-eight in Ambystoma tigrinum. 

2. Della Valle’s contention that variation in chromosome 
number is the rule is unconfirmed. 

3. The chromosomes form approximately a duplicate series of 
sizes and forms, supporting the contention that they consist of 
pairs of maternal and paternal homologues. 

4. An approximate constancy of size relations between pairs 
in the complexes of different individuals is also maintained. 

5. Della Valle’s claim that the chromosome lengths are a series 
of variants is not substantiated. 

6. There is evidence of unequal homologues in these cells. 

7. There is also a suggestion that there is a sex chromosome 
attached to a euchromosome. 


222 


CHARLES L. PARMENTER 


8. There is apparently but little complete fragmentation of 
the chromosomes. 

9. The above observations support the theory of the indi¬ 
viduality of the chromosomes. 

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CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 225 


Morris, Margaret 1914 The behavior of the chromatin in hybrids between 
Fundulus and Ctenolabrus. Jour. Exp. Zool., vol. 16. 

Mulsow, C. 1912 Der chromosomen cyclus bei Ancyracanthus cystidicola 
Rud. Arch. f. Zellf., Bd. 9. 

Rabl, C. 1885 tlber Zelltheilung. Morph. Jahrb., Bd. 10. 

1906 tlber organbildende Substanzen und ihre Bedeutung fur die 
Vererbung. Antrittsvorlesung. Leipzig. 

Richards, A. 1917 The history of the chromosomal vesicles in Fundulus and 
the theory of genetic continuity of the chromosomes. Biol. Bull., 
vol. 32. 

Robertson, W. R. B. 1915 Chromosome studies. III. Inequalities and de¬ 
ficiencies in homologous chromosomes; their bearing upon synapsis 
and the loss of unit characters. Jour. Morph., vol. 26. 

1916 Chromosome studies. I. Taxonomic relations shown in the 
chromosomes of Tettigidae and Acrididae: Y-shaped chromosomes and 
their significance in the Acrididae, Locustidae and Gryllidae: chromo¬ 
somes and variation. Jour. Morph., vol. 27. 

Schleip, W. 1911 Das Verhalten des Chromatins bei Angiostomum (Rhabdo- 
nema) nigrovenosum. Ein Beitrag zur Kenntniss der Beziehungen 
zwischen chromatin und Geschlechtsbestimung. Arch. f. Zellf., Bd. 7. 
Schreiner, A. and K. E. 1906 a Neue Studien iiber die Chromatinreifung der 
Geschlechtszellen. I. Die Reifung der mannlichen Geschlechtszellen 
von Tomopterus onisciformis. Arch, de Biol., Bd. 22. 

1906 b Ibid. II. Reifung der mannlichen Geschlechtszellen von 
Salamandra maculosa, Spinax niger and Myxine glutinosa. Ibid., 
Bd. 22. 

1908 Gibt es eine parallele Konjugation der Chromosomen? Erwid- 
erung an die Herren Fick, Goldschmidt und Meves. Videnskbs- 
Selskab. Schrifter I Math-Naturv. Klasse. No. 4. 

Seiler, J. 1913 Das Verhalten der Geschlechtschromosomen bei Lepidopteren. 

Nebst einem Beitrag zur Kenntniss die Eireifung, Samenreifung und 
Befruchtung. Arch. f. Zellf., Bd. 13. 

Snook, H. J., and Long, J. A. 1914 Parasynaptic stages in the testes of Aneides 
(Autodax) lugubris (Hallowell). Univ. Calif. Pub., vol. 15. 

Stevens, N. M. 1908 A study of the germ cells of certain Diptera, with 
reference to the heterochromosomes and the phenomena of synapsis. 
Jour. Exp. Zool., vol. 5. 

1910 The chromosomes in the germ cells of Culex. Jour. Exp. Zool., 
vol. 8. 

1911 Further studies on heterochromosomes in mosquitoes. Biol. 
Bull., vol. 20. 

Sutton, W. S. 1902 On the morphology of the chromosome group in Brachy- 
stola magna. Biol. Bull., vol. 4. 

Torok, L. 1888 Die Theilung der rothen Blutzellen bei Amphibien. Arch. f. 
mikr. Anat., Bd. 32. 

Wenrich, D. H. 1916 The spermatogenesis of Phrynotettix magnus, with 
special reference to synapsis and the individuality of the chromosomes. 
Bull. Museum of Comp. Zool., Harvard College, vol. 60. 


226 


CHARLES L. PARMENTER 


Wenrich, D. H. 1917 Synapsis and chromosome organization in Chorthippus 
(Stenobothrus) curtipennis and Trimerotropis suffusa (Orthoptera). 
Jour. Morph., vol. 29. 

Whiting, P. W. 1917 The chromosomes of the common house-mosquito, Culex 
pipiens. Jour. Morph., vol. 28. 

Wilson, E. B. 1906 Studies on chromosomes. III. The sexual differences of 
the chromosomes in Hemiptera, etc. Jour. Exp. Zool., vol. 3. 

1909 Studies on chromosomes. V. The chromosomes of Metapodius, 
etc. Ibid., vol. 6. 

1910 Studies on chromosomes. VI. A new type of chromosome com¬ 
bination in Metapodius. Ibid., vol. 9. 

1911 Studies on chromosomes. VII. A review of the chromosomes of 
Nezara, etc. Jour. Morph., vol. 22. 

1912 Studies on chromosomes. VIII. Observations on the matura¬ 
tion phenomena in certain Hemiptera, etc. Jour. Exp. Zool., vol. 13. 

Winiwarter, H. von 1900 Le corpuscle intermediare et le nombre des chromo¬ 
somes chez le Lapins. Arch, de Biol., T. 16. 

Woolsey, Carrie I. 1915 Linkage of chromosomes correlated with reduction 
in numbers among the species of a genus, also within a species of 
Locustidae. Biol. Bull., vol. 28. 


PLATES 


EXPLANATION OF PLATES 

The drawings were made with the aid of a camera lucida, using a Zeiss 2-mm. 
apochromatic immersion objective, N. A. 1.30, and a Spencer compensating 
ocular 20 X which produced a magnification of 2633. The illumination consisted 
of light from a 100-watt frosted-globe Mazda concentrated-filament lamp passed 
through a daylight glass or a common cobalt-blue glass filter, and an Abbe con¬ 
denser. The observer was shaded from the light in front of him and from the 
sides by a black-cloth screen. 

In reproduction plates 1 to 8 have been reduced one-third, and plate 9 three- 
fourths, giving a final magnification of 1755 and 1316, respectively. 

Figures 1 to 20 represent complexes of class I; figures 21 and 23, complexes of 
class II. 


PLATE 1 

EXPLANATION OF FIGURES 

1 to 3 Very late peritoneal prophases. 

4 A peritoneal metaphase. 

The numbered pairs of chromosomes correspond to pairs bearing, respectively, 
the same numbers in figures 27 and 28, and in the legend of figures 33 and 34. 


228 


PLATE 1 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 






229 





PLATE 2 

EXPLANATION OF FIGURES 

5 and 6 A gill-plate prophase and metaphase. 

7 and 8 Metaphases of the tail epithelium. 


230 


PLATE 2 



CHROMOSOME NUMBER’and PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTKR 


7 


231 


8 



PLATE 3 

EXPLANATION OF FIGURES 

9 to 12 Late prophase complexes of the lung epithelium. 

The pair numbers correspond Respectively, to those in figures 29^and 30, and 
in the legend of figures 35 and 36. 


232 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 


PLATE 3 



233 








































PLATE 4 

EXPLANATION OF FIGURES 

13 to 16 Gill-plate complexes. The chromatids are shown only in figure 13. 


234 


PLATE 4 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 




235 



















PLATE 5 

EXPLANATION OF FIGURES 

17 to 19 Two prophases and one early metaphase of gill-plate epithelium. 
20 A peritoneal metaphase. 


236 


PLATE 5 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 



237 























PLATE 6 

EXPLANATION OF FIGURES 

21 and 23 Gill-plate prophases of class II. Chromosomes ‘,/Y figure 21, inter¬ 
preted as one; ‘i,’ figure 23, as two (p. 12). 

22 A peritoneal complex with part of the chromosomes missing. 

24 A peritoneal complex separated into two parts. The pair numbers are 
duplicated in figure 31 and in the legend of figure 37. 


238 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 


PLATE 6 





239 


JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 














PLATE 7 

EXPLANATION OF FIGURES 

25A Another peritoneal complex separated into two parts which are drawn 
in their relative positions. X 866. 

25B The chromosomes of figure 25A. X 1755. 

Chromosomes 14 to 17 are interpreted; note that the chromatids of these four 
chromosomes are well separated. 

26A A lung epithelial cell separated into two parts which are drawn in their 
relative positions. X 866. 

26B Chromosomes of 26A. X 1755. Pair numbers duplicated in figure 32. 


240 


PLATE 7 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 




25 B 




241 










PLATE 8 

EXPLANATION OF FIGURES 

27 to 32 Chromosomes of figures 1, 3, 9, 10, 24, and 26, respectively, arranged 
in pairs with pair numbers and figures indicating their lengths in millimeters 
at X 2633. The amount included in any figure for foreshortening is indicated 
above that figure. The pair numbers are duplicated in the above figures and in 
the legend of figures 33 to 37, respectively. 


242 


CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 

CHARLES L. PARMENTER 


27 ,2.5 13 


15.1 16.2 





+.7 + 2.8 


27.6 276 



+.7 

30+ 33 






l% JV? 

31 8.5 10.5 14 ! 6-5 



20 20 




24 24 



25 26 



29 33.5 


32 6 8 II 14 

I 2 




17 17.5 


4 



19 19 


5 



19 20 


6 



22 23 26 26 


7 8 


243 


244 


PLATE 8 








39 41 




26 27 

9 



28 28 


10 



245 



































































































































PLATE 9 


EXPLANATION OF FIGURES 

33 to 37 Representing the lengths of the chromosomes in the cells indicated 
in the table below at a magnification of 1316. The differences in the lengths of 
the lines and also the spaces between the lines represent relative differences in 
chromosome lengths. For convenience the width of the spaces between the 
lines are made eight times the differences in lengths. The lengths of the chromo¬ 
somes and their percentage of the average length in the cell is sh.own in the 
table below. The amount of foreshortening in any chromosome is indicated in 
plate 8. 


FIGURES 


PAIRS 

33 (1, 27) 

34 (3 

, 28) 

35 (9, 29) 

36 (10, 30) 

37 (24, 31) 


Milli¬ 

metres 

Per 

cent 

Milli¬ 

metres 

Per 

cent 

Milli¬ 

metres 

Per 

cent 

Milli¬ 

metres 

Per 

cent 

Milli¬ 

metres 

Per 

cent 

' { 

12.5 

37 

14.0 

35 

18.0 

44 

14.5 

34 

8.5 

27 

13.0 

38 

14.5 

36 

18.5 

45 

17.5 

41 

10.5 

33 

2 1 

15.1 

44 

17.8 

45 

19.0 

46 

18.0 

42 

14.0 

44 

2 1 

16.2 

47 

17.9 

45 

19.5 

47 

20.0 

47 

16.5 

52 

3 ( 

22.5 

66 

25.5 

64 

26.7 

65 

24.7 

58 

20.0 

63 

3 1 

23.3 

68 

26.9 

68 

27.5 

68 

25.0 

59 

20.0 

63 

4 1 

26.5 

77 

27.8 

70 

29.0 

71 

27.0 

63 

22.0 

70 

4 l 

26.8 

78 

28.6 

72 

29.5 

72 

29.0 

68 

22.0 

70 

5 1 

27.2 

80 

31.6 

79 

31.0 

76 

31.0 

73 

23.5 

75 

5 i 

27.2 

80 

32.1 

80 

31.6 

77 

33.0 

78 

23.5 

75 

6 { 

27.6 

81 

30+ 

.— 

33.5 

82 

35.4 

84 

24.0 

76 

27.6 

81 

33.0 

83 

35.5 

87 

35.5 

84 

24.0 

76 

7 1 

28.4 

83 

33.6 

84 

36.5 

89 

36.5 

86 

25.0 

79 

7 l 

28.9 

84 

33.6 

84 

39.5 

97 

42.0 

99 

26.0 

81 

8 { 

36.0 

105 

42.0 

105 

40.4 

99 

44.8 

103 

29.0 

91 

36.0 

105 

42.0 

105 

46.0 

112 

44.8 

103 

33.5 

106 

9 { 

40.4 

118 

49.7 

125 

47.5 

116 

49.0 

116 

39.0 

124 

52.8 

154 

49.7 

125 

48.0 

117 

49.5 

117 

41.0 

130 

10 { 

43.0 

126 

50.7 

127 

50.0 

122 

54.0 

127 

41.0 

130 

44.0 

129 

52.3 

132 

54.0 

132 

55.0 

130 

41.0 

130 

14 { 

44.4 

130 

53.8 

135 

55.5 

136 

57.3 

135 

43.0 

136 

45.3 

132 

53.8 

135 

55.5 

136 

58.8 

138 

44.5 

141 

12 { 

45.7 

134 

55.4 

139 

56.5 

138 

60.0 

142 

44.5 

141 

45.8 

134 

56.8 

143 

56.5 

138 

61.3 

145 

54.0 

171 

13 { 

48.9 

143 

58.7 

148 

58.5 

143 

62.2 

147 

47.0 

149 

49.3 

144 

58.9 

148 

58.5 

143 

64.7 

153 

47.5 

150 

14 { 

51.0 

149 

62.2 

156 

60.0 

147 

66.3 

156 

48.0 

152 

52.0 

152 

64.0 

161 

62.0 

151 

69.3 

163 

49.5 

157 


248 

































PLATE 9 



CHROMOSOME NUMBERfAND PAIRS IN AMBYSTOMA 


CHARLES L. PARMENTBR 



249 





































































































































































































































































* 
























































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